Application of AAV overexpression of macrophage MCT1 in the treatment of corneal nerve injury repair in mice

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to application of a nucleic acid vector encoding a macrophage monocarboxylate transporter MCT1 in preparation of a pharmaceutical composition for promoting corneal nerve regeneration and recovery of corneal sensitivity after corneal nerve injury. The present application finds that after corneal nerve injury, increasing the level of macrophage MCT1 can drive the change of the metabolic mode of macrophages to the direction of enhanced oxidative phosphorylation and reduced glycolysis, reduce the expression of local pro-inflammatory related factors, and increase the level of pro-regeneration factors and neurotrophic factors, thereby improving the regeneration of corneal nerve structure and the recovery of sensory function. Therefore, a macrophage-targeted or macrophage-biased expression vector containing a nucleic acid sequence encoding MCT1 can be used for preparation of a pharmaceutical composition for treating corneal nerve injury.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of macrophage monocarboxylic acid transporter MCT1 in the repair of corneal nerve injury. Background Technology

[0002] Normal, intact corneal nerves are responsible for maintaining corneal sensation, blink reflex, and ocular surface homeostasis, and are an indispensable part of the cornea as a barrier against external stimuli. Neurotrophic keratopathy (NK) is a degenerative corneal disease that can lead to decreased or absent corneal sensation, corneal thinning, poor healing after corneal injury, persistent epithelial defects, corneal dissolution or perforation, and vision loss. Many systemic and local conditions, including surgery and topical medications, can cause NK, such as herpes zoster or herpes simplex keratitis, chemical burns, physical injuries, corneal surgery, prolonged use of contact lenses, or damage to the trigeminal nerve. The estimated prevalence of NK is 5 / 10000 (0.05%). The decreased blinking frequency and reduced tear production caused by decreased corneal sensitivity in NK can induce and maintain an inflammatory environment and perpetuate corneal epithelial lesions. Meanwhile, healthy corneal epithelium plays a role similar to Schwann cells in supporting exposed corneal nerves. Nerve fibers lacking epithelial support are easily damaged and become dysfunctional, leading to a vicious cycle of evaporation-overstimulation-neurogenic inflammation-epithelial damage-abnormal nerve activity-inflammation. In the pathogenesis of NK cell disease, reduced tear clearance leading to the accumulation of toxic substances and pro-inflammatory cytokines produced by local immune cells such as macrophages on the ocular surface is a significant contributing factor to its development.

[0003] Currently, clinical treatments for neurotrophic corneal lesions and corneal nerve injuries mainly include artificial tears, therapeutic contact lenses, amniotic membrane transplantation, autologous serum or platelet-rich plasma preparations, recombinant human nerve growth factor (cenegermin / rhNGF), and corneal neurotrophic surgery. While these methods can promote epithelial closure or provide neurotrophic support to some extent, they still suffer from problems such as frequent administration, limited applicability, high cost, surgical trauma, or insufficient regulation of the local inflammatory-immune metabolic microenvironment. Therefore, there is a clear clinical need to develop treatment strategies that can simultaneously improve the corneal nerve regeneration microenvironment and promote sensory function recovery.

[0004] The cornea possesses unique tissue characteristics, including being avascular, transparent, and subject to immune regulation. Its nerve regeneration process cannot be simply equated with the repair of ordinary peripheral nerves such as the sciatic nerve. Corneal epithelial cells, stromal corneal cells, trigeminal nerve endings, and resident or infiltrative myeloid cells collectively constitute the local neuroimmune microenvironment. Previous studies have shown that resident corneal macrophages can form a spatial proximity relationship with corneal nerve bundles; this relationship rapidly changes after aseptic injury to the corneal epithelium, suggesting that macrophages participate in the local response following corneal nerve injury.

[0005] The distribution of macrophages in the cornea is closely related to the anatomy of corneal nerves. Resident macrophages in the cornea surround large corneal nerve trunks at the periphery, and the number of these nerve-associated macrophages significantly decreases 2 hours after aseptic corneal injury, returning to baseline levels after 72 hours. This suggests that macrophages may participate in the corneal nerve injury response. Studies have found that 24-48 hours after aseptic corneal injury, damaged corneal cells release the small heat shock protein HSPB4 and activate resident macrophages through the TLR2 / NF-κB pathway. Following corneal scratching, stromal monocyte chemotactic activator (MCAF) expression induces monocyte-macrophage infiltration into the corneal stroma. In peripheral nerves, macrophages secrete cytokines, triggering the synthesis of growth factors in non-neuronal cells and producing factors that promote Schwann cell (SC) migration and axon regeneration. In recent years, many studies have found that macrophages also participate in the process of corneal nerve regeneration and repair. For example, in diabetic corneal neuropathy mice, treatment with PEDF+DHA promoted the recruitment of type 2 macrophages and accelerated the repair of corneal wounds and corneal nerves.

[0006] Monocarboxylic acid transporters (MCTs), particularly MCT1 (encoded by slc16a1), have been shown to regulate various immune cell functions. MCT1 is widely distributed and plays a role in lactate uptake. MCT1 is primarily located in the mitochondrial plasma membrane of macrophages. The absence of MCT1 in macrophages leads to altered macrophage metabolism, prompting the secretion of more pro-inflammatory factors. These alterations impair the repair process of peripheral nerve damage. However, the impact of altered macrophage metabolism in the cornea on corneal nerve regeneration remains unclear. Therefore, this study aims to explore the effects of altered macrophage metabolism on corneal nerve regeneration and the mechanisms underlying these effects.

[0007] On the other hand, damage-related molecules can activate local corneal inflammatory pathways such as TLR2 / NF-κB and IL-6, and excessive or persistent pro-inflammatory responses can exacerbate corneal nerve loss. However, specific myeloid cell subsets can also participate in neuroprotection by limiting inflammation amplification, clearing damage debris, and secreting pro-repair factors. Therefore, simply inhibiting inflammation cannot fully explain or resolve corneal nerve regeneration disorders. How to regulate the functional state of macrophages to a reparative state conducive to nerve regeneration is a technical problem that urgently needs to be solved.

[0008] Previous studies on non-ocular peripheral nerves have suggested that Slc16a1 / MCT1 is associated with macrophage metabolism, phagocytosis, and nerve repair. However, current techniques have not clarified the role of MCT1 in macrophages after corneal nerve injury, nor have they revealed whether increasing macrophage MCT1 levels can promote neural structure regeneration and corneal sensitivity recovery in this unique ocular surface tissue.

[0009] Therefore, exploring the effects of the monocarboxylic acid transporter MCT1 on macrophage metabolism, its influence on corneal nerve repair, and the mechanisms by which these effects occur will help promote corneal nerve function repair and provide a theoretical basis for clinical treatment.

[0010] This invention clarifies that the monocarboxylic acid transporter MCT1 plays a regulatory role in pro-inflammatory factors, regenerative factors, and neurotrophic factors during the corneal nerve injury repair process. By increasing the level of the monocarboxylic acid transporter MCT1, the local inflammatory microenvironment can be effectively regulated, thereby promoting the repair of corneal nerve injury. Summary of the Invention

[0011] This invention demonstrates that increasing the level of the macrophage monocarboxylic acid transporter MCT1 plays a positive regulatory role in the repair of corneal nerve injury. Specifically, by regulating the metabolic pattern of macrophages and the local inflammatory microenvironment of the cornea, it reduces the expression of pro-inflammatory factors, increases the levels of regenerative factors and neurotrophic factors, promotes the regeneration of corneal nerve structures and the recovery of corneal sensitivity, thereby demonstrating that the nucleic acid carrier encoding MCT1 can serve as a technical means for preparing drug compositions for the treatment of corneal nerve injury.

[0012] To achieve the above-mentioned objectives, the technical solution provided by this invention is as follows: This invention provides the use of a nucleic acid vector encoding the macrophage monocarboxylic acid transporter MCT1 in the preparation of a pharmaceutical composition for promoting corneal nerve regeneration and recovery of corneal sensitivity after corneal nerve injury.

[0013] In this invention, the nucleic acid vector is a macrophage-targeting or macrophage-biased expression vector that can enhance the expression level of MCT1 in macrophages.

[0014] In this invention, the pharmaceutical composition is used to promote corneal nerve regeneration and recovery of corneal sensitivity.

[0015] In this invention, the nucleic acid vector promotes corneal nerve regeneration and corneal sensitivity recovery by increasing macrophage MCT1 levels, enhancing macrophage oxidative phosphorylation levels, reducing glycolysis levels, limiting the expression of IL-1β and / or Lyz1 pro-inflammatory factors, and increasing the levels of Arg1, Ym1, NGF and / or CNTF pro-regeneration or neurotrophic factors.

[0016] In this invention, the nucleic acid vector contains a promoter and a nucleic acid sequence encoding MCT1, wherein the promoter is the macrophage-specific promoter F4 / 80 or a promoter capable of driving expression in macrophages.

[0017] In this invention, the nucleic acid vector is an adeno-associated virus vector, preferably an AAV2 / 9 vector.

[0018] In this invention, the nucleic acid sequence encoding MCT1 includes the mouse MCT1 coding sequence shown in SEQ ID NO:1.

[0019] In this invention, the pharmaceutical composition is a formulation suitable for ophthalmic administration, subconjunctival administration, periorbital administration, or intravenous administration; in mouse animal experiments, the adeno-associated virus vector can be delivered via injection into the posterior orbital sinus.

[0020] According to the present invention, increasing the level of the macrophage monocarboxylic acid transporter MCT1 can regulate macrophage metabolic patterns, enhance oxidative phosphorylation, inhibit glycolysis, and modulate the local inflammatory microenvironment of the cornea, thereby promoting structural regeneration and sensory function recovery after corneal nerve injury. This technical solution helps to understand the corneal nerve injury repair process from the perspective of neuro-immune-metabolic regulation and provides a new intervention strategy for corneal nerve injury and neurotrophic corneal lesions. Attached Figure Description

[0021] In the following description of the accompanying drawings and embodiments, for convenience, the term "Neurotrophic Keratopathy" will be abbreviated as "NK"; the term "Adeno-associated Virus" will be abbreviated as "AAV"; the term "Bone Marrow-Derived Macrophages" will be abbreviated as "BMDMs"; the term "Extracellular Acidification Rate" will be abbreviated as "ECAR"; the term "Oxygen Consumption Rate" will be abbreviated as "OCR"; and the term "Monocarboxylate Transporter 1" will be abbreviated as "MCT1".

[0022] Appendix Figure 1-5 This is a schematic diagram illustrating the upregulation of MCT1 expression and inflammatory cytokine secretion in corneal macrophages during corneal abrasion, according to an embodiment of the present invention. Appendix Figure 6-9 This is a schematic diagram illustrating the reverse metabolic reprogramming induced by macrophage MCT1 overexpression to promote corneal nerve regeneration according to an embodiment of the present invention. Appendix Figure 10-12 This is a schematic diagram illustrating that macrophage-specific knockout of the slc16a1 gene does not affect corneal epithelial damage repair, but inhibits corneal nerve regeneration after injury, according to another embodiment of the present invention. Appendix Figure 13-18This is a schematic diagram illustrating how macrophage-specific knockout of the slc16a1 gene aggravates the expression of inflammatory factors, inhibits the expression of regeneration factors, and promotes macrophage infiltration after corneal injury, according to another embodiment of the present invention. Appendix Figure 19-21 This is a schematic diagram illustrating how macrophage-specific knockout of the slc16a1 gene affects corneal nerve regeneration by regulating the expression of neurotrophic factors, according to an embodiment of the present invention. Appendix Figure 22-24 This is a schematic diagram illustrating how macrophage-specific knockout of the slc16a1 gene can alter macrophage cellular energy metabolism and inflammatory cytokine expression in vitro, and inhibit macrophage phagocytic activity in vitro, according to an embodiment of the present invention. Appendix Figure 25-26 This is a schematic diagram illustrating the effect of macrophages with the slc16a1 gene specifically knocked out according to an embodiment of the present invention on the axonal growth of trigeminal neurons in vitro. Appendix Figure 27-29 This is a schematic diagram illustrating how subconjunctival injection of macrophages with intact MCT1 function can improve corneal nerve regeneration delay in macrophage-specific MCT1-deficient mice according to an embodiment of the present invention. Detailed Implementation

[0023] The invention will be described in detail below with reference to exemplary embodiments, but the invention is not limited to these embodiments. The invention is embodied in various forms described below, but should not be construed as limited to the exemplary embodiments set forth herein. Therefore, the detailed description and embodiments of the invention will convey the scope of the invention to those skilled in the art and are to be construed as falling within the scope of the invention.

[0024] Example 1: Animal experiments showed that overexpression of macrophage MCT1 can significantly promote corneal nerve regeneration and recovery of corneal sensitivity. 1. Experimental Methods

[0025] (1) Construction of a mouse corneal injury model C57BL / 6J mice were used to establish a mouse model of corneal injury. Mice were anesthetized with 5% chloral hydrate. Under a stereomicroscope, a 3 mm lesion was created in the central corneal epithelium using Algerbrush II corneal rust ring remover (Alger Co, Lago Vista, TX). After modeling, antibiotic eye ointment (levofloxacin eye ointment) was applied to prevent infection. The healing process of the corneal epithelial wound was evaluated by sodium fluorescein staining under a slit lamp. This method is a classic approach for constructing a mouse model of corneal nerve injury, a common cause of corneal nerve injury that can damage the structure and function of the cornea and corneal nerves.

[0026] (2) Inject the drug into the posterior orbital sinus of mice.

[0027] Adeno-associated virus vectors (AAV-F4 / 80-MCT1) carrying nucleic acid translated into MCT1 via macrophage-specific promoters and empty adeno-associated virus vectors (HAAV2 / 9-F4 / 80-NC) were used to upregulate the expression levels of the slc16a1 gene and MCT1 protein in mouse macrophages via retroorbital sinus injection. Specifically, mice were fixed with their eyeballs protruding from the orbit. Using an insulin syringe, the needle was inserted at a 90° angle to the center of the mouse's orbit to a depth of 2-3 mm. Injection was performed upon observation of blood return. The same number of mice were injected via retroorbital sinus injection with 100 μL of a 1×10⁻⁶ titer. 12 VG / mL AAV-NC served as the negative control. The AAV-F4 / 80-MCT1 overexpression vector and the HAAV2 / 9-F4 / 80-NC control vector were purchased from Shanghai Hanheng Gene Technology Co., Ltd. The basic structure of AAV-F4 / 80-MCT1 is HBAAV2 / 9-F4 / 80-m-Slc16a1-Null-ZsGreen, and the basic structure of HAAV2 / 9-F4 / 80-NC is HBAAV2 / 9-F4 / 80-ZsGreen.

[0028] (3) Corneal fluorescein staining Immediately after corneal epithelial scraping and modeling, and at 24h, 48h, and 72h, the ocular surface of mice was stained with sodium fluorescein solution. The staining was observed and photographed using a slit-lamp microscope with a cobalt blue filter, and the positive area of ​​sodium fluorescein staining was analyzed and statistically analyzed.

[0029] (4) Corneal sensitivity test Corneal sensitivity was measured in unanesthetized mice using a Cochet-Bonnet tactile meter (Luneau Ophtalmologie, Cedex, France). The test began with the maximum length of the nylon filament (6 cm) and was then reduced by 0.5 cm until the corneal contact threshold was reached. The longest nylon filament length that elicited a positive response was considered the threshold, and each result was validated three times.

[0030] (5) Isolation and culture of peritoneal macrophages 1 ml of 3% sterile mercaptoacetate (Sunshine Yingrui) was injected intraperitoneally into mice. Four days later, peritoneal exudate cells were collected by irrigating with PBS. 1 × 10⁻⁶ cells were then added to the peritoneum. 6 Aliquots of cells were seeded into 6-well culture plates and incubated at 37°C and 5% CO2 for 3 hours to allow them to adhere. Unattached cells were removed by vigorous washing three times with PBS and then incubated for another 24 hours under the same conditions before processing or analysis.

[0031] (6) Hippocampal bioenergy analysis Mature BMDMs cultured from macrophage-specific MCT1 knockout mice and littermate control mice, along with peritoneal macrophages from mice three weeks after injection of AAV virus overexpression and AAV control virus, were plated into Seahorse XF96 cell culture microplates. Oxygen consumption and extracellular acidification were measured using the XF 96 extracellular flux analyzer (SeahorseBioscience) according to the manufacturer's instructions. Macrophages were seeded at 20,000 cells / well in SeahorseXF 96 cell culture microplates and cultured for 24 hours in DMEM and F12 medium containing 5.5 mM d-glucose. Bioenergetic analysis was performed by sequential injection of 2 μM oligomycin, 4 μM FCCP, 0.5 μM rotenone, and 4 μM antimycin. ATP production from oxidative metabolism and glycolysis was calculated using formulas. Data are expressed as OCR (picomoles per minute, pmol / min) and ECAR (milli-pH per minute, mpH / min) for 20,000 cells.

[0032] (7) Immunofluorescence staining of mouse whole cornea slides, cell immunofluorescence staining and frozen section immunofluorescence staining On days 7 and 14 following corneal epithelial curettage, mouse eyeballs were harvested and fixed on ice in Zamboni fixative for 2 hours. The cornea was then dissected around the sclera-limbus region. The cornea was blocked at room temperature for 2 hours in phosphate-buffered saline containing 0.1% Triton X-100, 2% goat serum, and 2% bovine serum albumin. It was then incubated overnight in Tris-buffered saline containing an Alexa Fluor 488-binding neuronal class III beta-tubulin antibody (1:100, 657404, Biolegend), 0.1% Triton X-100, 2% goat serum, and 2% bovine serum albumin. After cleaning the cornea six times, it was cut into four flaps and observed under a fluorescence microscope. Image J version 1.54p software was used to calculate the coverage of corneal nerves per unit area of ​​corneal epithelium.

[0033] Eyeballs from mice in the experimental and control groups were sectioned using frozen sections or 96-well ultrathin low-transparency black plates containing trigeminal neuron cell crawling sections after co-culture and macrophages after phagocytosis experiments. Sections were fixed in 4% paraformaldehyde solution for 10 minutes, then immersed in 0.2% Triton X-100 solution for 3 minutes. After blocking with goat serum at room temperature for 30 minutes, the sections were inoculated with primary antibodies MCT1 (20139-1-AP, Proteintech), F4 / 80 (14-4801-82, Invitrogen), Lyz-1 (ab108508, Abcam), Egr-1 (55117-1-AP, Proteintech), CD68 (ab283654, Abcam), NGF (sc-32300, Santa Cruz Biotechnology), and CNTF (ab270992). Abcam and βⅢ-Tubelin (657404, Biolegend) were incubated overnight at 4°C. After washing three times with PBS, the sections were incubated with fluorescein-conjugated secondary antibody at room temperature for 2 hours. A mounting medium containing DAPI (ab104139, Abcam) was dropped onto the sample and covered with a coverslip. The sections were observed and photographed under a confocal microscope.

[0034] (8) qRT-PCR Total RNA was isolated from the samples using Trizol and resuspended in DEPC water. The obtained RNA was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). qRT-PCR was performed using iTaq Universal SYBR Green Supermix reagents (Bio-Rad) on a CFX96 PCR system. Sample mRNA expression levels were determined using... express. 2. Experimental Results

[0035] (1) After corneal nerve injury, the expression of MCT1 and related inflammatory factors in corneal macrophages increases. Appendix Figure 1 Figure A above shows a 3mm diameter corneal central epithelial scraping model. (Attached) Figure 1 Figure A below shows the results of sodium fluorescein staining 72 hours after modeling, confirming that the corneal epithelium has been completely repaired, with no positive staining signal observed, indicating that the corneal epithelial repair process after injury is normal.

[0036] Appendix Figure 1 B and appendix Figure 1C shows the results of MCT1 immunofluorescence staining, indicating widespread expression of MCT1 in both the corneal epithelium and endothelium. On day 3 post-injury (D3), the expression levels of MCT1 (green fluorescence) in the corneal epithelium, endothelium, and stroma were significantly higher than before injury and on day 1 post-injury. (See attached image.) Figure 2 A. Appendix Figure 2 B shows the results of immunofluorescence double staining of F4 / 80 (green fluorescence) and MCT1 (red fluorescence). On day 3 post-injury, the expression level of MCT1 in corneal infiltrating macrophages was significantly increased.

[0037] Appendix Figure 3 A and Appendix Figure 3 B shows the results of gene expression detection in corneal tissue. The mRNA expression levels of MCT1 and IL-1β, markers of M1 macrophages, peaked on day 3 post-injury and then declined until day 7. Figure 3 C shows that the relative mRNA expression level of CD206, a marker of M2 macrophages, was persistently upregulated on both day 3 and day 7 post-injury.

[0038] Appendix Figure 4 A shows the cell annotation and cluster analysis completed in this study based on the corneal injury-related single-cell transcriptome sequencing dataset GSE171830; Appendix Figure 4 B shows the analysis results of the GSE247392 dataset. 24 hours after corneal injury, the mRNA expression level of MCT1 in corneal macrophages was significantly upregulated, and the upregulation was most significant in corneal immune cells.

[0039] Appendix Figure 5 A illustrates that, based on cell surface markers and gene expression profiles, this invention further divides corneal macrophages into three subgroups: corneal resident macrophages... monocytes and inflammatory macrophages derived from monocytes Appendix Figure 5 B shows that the mRNA expression level of MCT1 in SC3 subpopulation macrophages is significantly higher than that in the other two subpopulations.

[0040] In summary, the above results indicate that macrophages and their secreted cytokines are involved in the inflammatory response and repair and regeneration process following corneal nerve injury, and macrophage MCT1 likely plays a key regulatory role in this process.

[0041] (2) Overexpression of MCT1 in mouse macrophages can accelerate the regeneration process after corneal nerve injury, and the oxidative phosphorylation level of macrophages is significantly increased. A mouse model of macrophage-specific MCT1 overexpression was constructed using an adeno-associated virus type 2 / 9 (AAV2 / 9) vector. The vector was designed to drive the expression of the MCT1-encoding gene Slc16a1 using the macrophage-specific promoter F4 / 80, while also carrying the ZsGreen reporter gene. Figure 6 This paper presents the experimental timeline for the introduction of the vector into C57BL / 6J mice via retroorbital sinus injection. After the virus was stably expressed in the mice, a 3 mm diameter central corneal epithelial scraping was performed, and corneal nerve regeneration was assessed on day 7 (D7) and day 14 (D14) post-injury. To verify the overexpression efficiency of MCT1, mouse peritoneal exudate macrophages were isolated, and gene expression levels were detected using real-time quantitative polymerase chain reaction (qRT-PCR). (Appendix) Figure 7 A shows that, compared with mice injected with the control virus (AAV-CON group), mice injected with the overexpressing virus (AAV-OE group) had a significantly higher relative expression level of MCT1 mRNA in their macrophages, confirming the successful construction of the MCT1 overexpression model.

[0042] After successful validation of the overexpression model, this invention constructed a corneal epithelial scraping model, and tested the corneal sensitivity of mice on days 7 and 14 post-injury. Figure 7 B shows that at both time points, the corneal sensitivity recovery in the AAV-OE group was significantly better than that in the AAV-CON group (D7, AAV-OE group vs AAV-CON group = 50±8.944mm vs 45±8.944mm; D14, AAV-OE group vs AAV-CON group = 60mm vs 57.5±2.739mm); and whole-corneal βⅢ microtubule immunofluorescence staining was performed, with the attached... Figure 7 E indicates the state of corneal nerve regeneration. Figure 7 C and D show that the corneal nerve density and the number of nerve branch points in the AAV-OE group mice were significantly higher than those in the AAV-CON group at both time points D7 and D14.

[0043] The above results indicate that MCT1 is not only an essential key molecule in corneal nerve regeneration, but also that specific upregulation of MCT1 expression in macrophages can accelerate the nerve regeneration process, suggesting that MCT1 may be a potential therapeutic target for corneal nerve injury.

[0044] To elucidate the underlying molecular mechanism, this invention collected mouse corneal tissue on day 3 after injury and used qRT-PCR technology to detect the mRNA expression levels of pro-inflammatory factors, pro-regenerative factors, and ciliary neurotrophic factor (CNTF)—the expression levels of the above factors all showed significant changes in the corneal injury model of macrophage MCT1-deficient mice. Figure 8 A, B, and C show that the mRNA expression levels of IL-1β, Lyz-1, and TNFα in the corneal tissue of mice in the AAV-OE group were significantly lower than those in the AAV-CON group. Figure 8 Figures D and E show that the mRNA expression levels of Arg-1 and CNTF were significantly higher in the AAV-CON group than in the AAV-CON group. This suggests that the mechanism by which macrophage MCT1 overexpression promotes corneal neurogenesis is at least partly achieved through the upregulation of regeneration-promoting factors and key neurotrophic factors.

[0045] Appendix Figure 9 AG illustrates the use of a hippocampal extracellular flux analyzer to detect the metabolic function of mouse peritoneal exudate macrophages, clarifying the effect of MCT1 overexpression on macrophage metabolic patterns. Figure 9 A, F, and G show the results of extracellular acidification rate (ECAR) detection. The glycolytic level and glycolytic capacity of macrophages in the AAV-OE group were significantly lower than those in the AAV-CON group. Figure 9 D, C and Figure 9 E shows the results of oxygen consumption rate (OCR) detection. The basal respiratory oxygen consumption rate, FCCP-induced maximum respiratory oxygen consumption rate, and reserve respiratory capacity of macrophages in the AAV-OE group were all significantly increased.

[0046] The aforementioned metabolic characteristics indicate that macrophage-specific MCT1 overexpression can drive a shift in cellular metabolic patterns towards an oxidative phenotype, specifically manifested as enhanced oxidative phosphorylation and suppressed glycolysis. This metabolic reprogramming process likely lays a crucial foundation in energy metabolism and cellular function for the accelerated corneal nerve regeneration observed in the experiments. Example 2: In vivo and in vitro experiments using macrophage MCT1-specific knockout mice demonstrated that macrophage MCT1 deficiency inhibits corneal nerve regeneration.

[0047] 1. Experimental Methods (1) Isolation and culture of BMDMs cells, isolation and culture of peritoneal macrophages and subconjunctival injection Bone marrow cells were prepared by washing bone marrow with cold DMEMF / F12-10 medium containing 10% FBS and 1% penicillin / streptomycin from macrophage-specific MCT1 knockout mice, littermate control mice, and wild-type mice. Cells were then incubated at 37°C in 5% CO2 gas in medium containing 20 ng / ml M-CSF, 10% FBS, and 1% penicillin / streptomycin DMEMF / F12-10. On day 4, non-adhesive cells were removed and culture medium was replenished. Subsequent detection or experiments were performed on day 7. For subconjunctival macrophage injection, wild-type mouse BMDMs were extracted 7 days prior to injection. After maturation, cell phenotype was identified by flow cytometry using F4 / 80 antibody and CD11b antibody.

[0048] Mature wild-type mouse BMDM cells were cultured, and BMDM was dissociated using a Cellstripper (Mediatech) and dispersed in PBS. Three days after corneal epithelial scraping, BMDM (5 × 10⁻⁶ cells / mL) was injected subconjunctivally using a microsyringe. 3 Administer MCT1 knockout mice or wild-type mice with 10 μL / eye cells per eye.

[0049] (2) Isolation and culture of trigeminal neuron cells and their co-culture with BMDMs cells After dissecting and exposing the trigeminal nerve of wild-type mice, remove bloodstains with a sterile cotton swab moistened with DMEMF / F12-10 medium containing 10% FBS and 1% penicillin / streptomycin. Cut and separate the trigeminal nerve, placing it in a culture dish containing the aforementioned DMEMF / F12-10 complete medium. After removing bloodstains, transfer it to another culture dish containing DMEMF / F12-10 complete medium. Cut the TN into 10-12 continuous portions, then digest with papain and collagenase for 20 minutes each, shaking to mix during digestion. After digestion, resuspend in L15 medium containing 5% FBS and 1% penicillin / streptomycin, carefully agitating with a pipette tip 40-50 times until no fragments remain, avoiding the introduction of air bubbles. Establish a Percoll gradient, add the cell suspension to the Percoll gradient, centrifuge at 2000 rpm for 35 min, remove the supernatant, resuspend the pellet in Neurobasal-A medium containing 2% B-27 additive and 1% glutamine additive, mix well, centrifuge again at 700 rpm for 8 min, aspirate the supernatant, add the medium again, mix well again, and then add the pellet to a six-well plate with a slide pre-coated with poly-L-lysine. Incubate the cells at 37°C in 5% CO2 atmosphere for 6 hours until they are fully adhered.

[0050] Seven days prior to the extraction of trigeminal neuron cells, BMDMs were extracted from macrophage-specific MCT1 knockout mice and littermate control mice and cultured to maturity. One day before co-culture, the mature BMDMs were dissociated, resuspended, and re-coated onto the upper chambers of a six-well transwell. On the day of co-culture, the transwell chambers containing well-adhered BMDMs were transferred to the upper chambers of the six-well plate containing trigeminal neuron cells. After co-culturing at 37°C in 5% CO2 gas for 24 h and 48 h, samples were taken to analyze the growth of trigeminal neuron cells.

[0051] (3) ELISA detection of supernatant protein content in co-cultured cell system Collect the supernatant of the culture medium after 48 hours of co-culture, centrifuge at 1000g for 20 minutes, and use the supernatant to detect the content of Egr-1 and Arg-1 proteins using an ELISA kit according to the instructions. Set up standard wells and sample wells, and set up 3 counting replicate wells for each sample. After incubation at 37℃, read the OD value using a microplate reader, calculate the concentration value of each sample, and perform statistical analysis.

[0052] (4) mRNA-sequencing analysis Total RNA was extracted from the samples using the SMART-Seq® HTKit kit. A cDNA library was constructed using the TruSeq® RNA Sample Preparation Kit. Specifically, the purified total RNA underwent polyA-containing RNA enrichment, fragmentation, first-strand cDNA synthesis, second-strand cDNA synthesis, end repair, 3' end A base addition, purification, and enrichment to ultimately construct the sequencing library. Quantification was performed using a Qubit® 2.0 fluorometer, and validation was performed using an Agilent 2100 bioanalyzer to determine the library's quality and size. Sequencing was performed using the Illumina HiSeq X-ten platform.

[0053] The raw data were preprocessed to remove rRNA sequences, primers, and low-quality data. Hisat2 (version 2.0.4) was used for genome matching, and StringTie (version 1.3.0) was used to count the number of fragments for each gene after matching, calculating the FPKM value for each gene. Differential gene analysis was performed between samples using edgeR, with thresholds set as FDR ≤ 0.05 and fold change ≥ 2. Differential genes were annotated using GO enrichment analysis and KEGG pathway enrichment analysis.

[0054] (5) Macrophage phagocytosis experiment Mature bone marrow-derived macrophages, isolated from macrophage-specific MCT1 knockout mice and littermate control mice, were cultured and seeded at approximately 70% confluence on 96-well ultrathin-bottom transparent black plates overnight until complete adherence. 2 μL of microspheres (1 μm, deep red fluorescence; Life Technologies F-8816) were suspended in 100 μL of PBS containing 1 mg / mL bovine serum albumin. Then, 20 μL was added to each well. The microspheres were allowed to settle onto the cells at 37°C and 5% CO2 for 2 hours. The culture medium was removed, and the cells were gently washed three times with PBS, then fixed with 4% PFA for 15 minutes. Macrophages were stained with anti-CD68 antibody (ab283654, Abcam, 1:200), and cell nuclei were stained with DAPI reagent (ab104139, Abcam). High-content microscopy analysis was performed to visualize and count the number of cells with internalized fluorescent microspheres, expressed as a percentage.

[0055] 2. Experimental Results 1) Macrophage-specific knockout of MCT1 does not affect corneal epithelial repair in mice, but it significantly impairs the regeneration of corneal nerves after injury. Appendix Figure 10 Appendix Figure 11 A illustrates the macrophage MCT1 gene conditional knockout mouse used in this invention, derived from the MCT1-Floxp tool mouse (MCT1). fl / fl MCT1 was obtained by crossing mice with myeloid cell-specific Cre tool mice (LysM-Cre), and genotyping was completed at 7-8 weeks of age. Bone marrow-derived macrophages (BMDMs) were isolated from both genotype mice, and their expression levels were detected after in vitro maturation. (See attached image) Figure 11 D shows that, compared with the control group mice (MCT1) fl / fl Compared to macrophage-specific MCT1 knockout mice (LysM-Cre MCT1), fl / fl MCT1 expression levels were significantly reduced in bone marrow macrophages derived from [a specific source], while [the expression level was significantly reduced in the appendix]. Figure 11 E showed that there was no significant difference in the mRNA expression level of MCT4 between the two groups of cells.

[0056] Appendix Figure 11 B shows the results of corneal immunofluorescence staining patch, indicating no significant difference in baseline corneal neural density between the two genotypes of mice (LysM-Cre MCT1). fl / fl Group: 15.46%~19.15% vs MCT1 fl / fl (Group: 16.56%~18.92%), suggesting that the absence of MCT1 in macrophages does not interfere with the development of corneal nerves in mice.

[0057] Appendix Figure 11C and F illustrate the present invention's assessment of epithelial repair progress at 0, 24, and 72 hours after corneal abrasion. The results show that... The corneal epithelial defect area of ​​the mice in the group (24h: 53.170%±7.222%; 48h: 27.463%±4.984%) was different from that in the control group. There was no significant difference in mice (24h: 55.238%±4.385%; 48h: 27.467%±5.911%), indicating that the loss of MCT1 in macrophages does not affect the corneal epithelial damage repair function.

[0058] Appendix Figure 12 A shows the results of immunofluorescence staining and imaging of corneal nerves on day 7 (D7) and day 14 (D14) after corneal abrasion, using βⅢ microtubule protein antibody, to further investigate the effect of macrophage MCT1 deficiency on corneal nerve regeneration. (Attached) Figure 12 B shows LysM-Cre MCT1 fl / fl The central corneal nerve density of the mice in the group (D7: 1.60%~2.11%; D14: 2.09%~3.54%) was significantly lower than that in the MCT1 group. fl / fl Group mice (D7: 2.19%~3.07%; D14: 3.56%~5.85%). Consistent with this, see attached... Figure 12 C shows LysM-Cre MCT1 fl / fl The number of nerve branch points in the central corneal region of the mice in the group (D7: 278-531; D14: 381-832) was also significantly less than that in the MCT1 group. fl / fl Group mice (D7: 408–537 mice; D14: 1666–2123 mice). Additionally, [the following is included] Figure 12 D shows LysM-Cre MCT1 fl / fl The corneal central sensitivity of the mice in the group (D7: 45±8.944mm; D14: 50±8.944mm) was significantly lower than that in the MCT1 group. fl / fl Group of mice (D7: 57.50±2.739mm; D14: 60mm).

[0059] In summary, the above results indicate that macrophage-specific MCT1 deficiency severely hinders the regeneration process of corneal nerves after injury.

[0060] (2) Macrophage-specific MCT1 knockout aggravates the expression of inflammatory factors at the site of corneal abrasion, inhibits the expression of pro-regeneration factors, and promotes macrophage infiltration, thereby hindering corneal nerve regeneration by regulating the expression of neurotrophic factors. To elucidate the molecular mechanism by which macrophage-specific MCT1 deficiency inhibits corneal nerve regeneration, this invention collected corneal tissue from two groups of mice on day 14 post-injury (D14) for transcriptome sequencing (mRNA-seq). Figure 13 B shows the analysis results, compared with the control group mice. Compared to macrophage-specific MCT1 knockout mice There are 344 differentially expressed genes (DEGs) in corneal tissue, of which 79 genes are upregulated and 265 genes are downregulated. (See attached image) Figure 13 A shows the results of enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG). These differentially expressed genes are mainly enriched in inflammatory response-related pathways, with the highest enrichment levels observed in the myocardial contractility pathway and the interleukin-17 (IL-17) signaling pathway. To clarify whether macrophage-specific MCT1 deficiency affects macrophage infiltration after corneal nerve injury, this invention involves F4 / 80 immunofluorescence staining of frozen eye sections, with attached... Figure 14 The study showed that on day 3 (D3) post-injury, there was no significant difference in the degree of macrophage infiltration in the corneal tissue between the two groups of mice; however, the appendix... Figure 15 A and B show LysM-Cre MCT1 up to day 7 post-injury (D7). fl / fl The number of macrophages infiltrating the corneal tissue of the group mice was significantly higher than that of the control group.

[0061] Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate differentially expressed genes related to the IL-17 pathway and macrophage phenotype in corneal tissue. The experimental time points were selected as follows: day 3 post-injury was the peak period for the release of pro-inflammatory cytokines from circulating macrophages; day 7 was the transition point where the peak release of pro-inflammatory cytokines subsided and the secretion of pro-regenerative factors began; and day 10 was the peak period for the release of pro-regenerative cytokines from circulating macrophages. (Appendix) Figure 16 A and B show the test results. On the 3rd day after the injury, LysM-Cre MCT1... fl / fl The mRNA expression levels of chemokine ligand 2 (CCL2) and chemokine ligand 7 (CCL7) in the corneal tissue of the LysM-Cre group mice were significantly higher than those in the control group. These results reveal that LysM-Cre MCT1... fl / fl The molecular mechanism by which increased macrophage infiltration occurred in the corneal tissue of mice on day 7 after injury.

[0062] Appendix Figure 16 C shows that on day 3 post-injury, the mRNA expression level of the pro-inflammatory factor lysozyme 1 (Lyz1) in the corneal tissue of the knockout group mice was significantly upregulated compared with the control group; Appendix Figure 16 D shows LysM-Cre MCT1 on day 7 post-injury. fl / flThe mRNA expression level of IL-1β in the corneal tissue of the group of mice was significantly increased. Figure 17 A shows that the mRNA expression level of tumor necrosis factor-α (TNF-α) was not significantly different from that of the control group on days 3 and 7 after injury. (See attached image.) Figure 16 F shows LysM-Cre MCT1 on day 10 post-injury. fl / fl The mRNA expression level of early growth response protein 1 (EGR1) in the corneal tissue of the control group was significantly higher than that of the control group.

[0063] Simultaneously, the expression levels of chitinase-like protein 3 (Ym1) and arginase 1 (Arg1) were detected; both are specific markers of alternative activation (M2 polarization) in mouse macrophages. (Appendix) Figure 16 E shows that on day 10 post-injury, LysM-Cre MCT1 fl / fl The mRNA expression level of chitinase-like protein 3 (Ym1) in the corneal tissue of the control group was significantly lower than that in the control group. Figure 17 B shows that the mRNA expression level of Arg-1 was not significantly different from that of the control group.

[0064] To clarify the protein expression levels of inflammatory factors in corneal tissue at different injury time points, this study conducted immunofluorescence staining experiments. (See attached image) Figure 15 A and C show LysM-Cre MCT1 on day 7 post-injury. fl / fl The relative fluorescence intensity of Lyz-1 in the corneal tissue of the control group was significantly higher than that of the control group. Figure 18 AB shows LysM-Cre MCT1 on day 10 post-injury. fl / fl The relative fluorescence intensity of EGR1 in the corneal tissue of mice in the group was significantly increased.

[0065] The above results suggest that after corneal nerve injury, In mice, upregulating the expression of CCL2 and CCL7 recruited more macrophages. Locally infiltrated macrophages were activated and highly expressed Lyz1, while the upregulated CCL7 further promoted macrophage polarization towards a pro-inflammatory phenotype (M1 type). This process led to further upregulation of pro-inflammatory factor (IL-1β) expression and activation of the downstream molecule EGR1, ultimately hindering corneal nerve regeneration by amplifying the inflammatory response. In short, the loss of macrophage-specific MCT1 in mice resulted in increased production of pro-inflammatory factors, decreased expression of regenerative factors, prolonged upregulation of inflammatory factors, and persistent activation of EGR1, ultimately inducing chronic corneal inflammation and inhibiting nerve regeneration.

[0066] Given that neurotrophic factors play a core regulatory role in corneal nerve regeneration, this study further investigated the effect of macrophage MCT1 deficiency on the expression of neurotrophic factors (NGF, CNTF, BDNF, PEDF) in damaged corneal tissue. mRNA expression levels were detected by qRT-PCR on day 3 post-injury. Figure 19 A shows LysM-Cre MCT1 fl / fl The mRNA expression level of nerve growth factor (NGF) in the corneal tissue of the group mice was significantly lower than that in the control group. Figure 20 A showed that the mRNA expression level of ciliary neurotrophic factor (CNTF) was not significantly different from that of the control group; on day 7 post-injury, the appendix Figure 19 B shows LysM-CreMCT1 fl / fl The mRNA expression level of CNTF in the corneal tissue of the group of mice was significantly reduced, while the expression level of the epidermal tissue was significantly reduced. Figure 20 B shows that the mRNA expression level of NGF was not significantly different from that of the control group. (See attached image) Figure 20 The mRNA expression levels of CD pigment epithelial-derived factor (PEDF) and brain-derived neurotrophic factor (BDNF) were not significantly different from those of the control group on days 3 and 7 after injury.

[0067] Appendix Figure 21 Figures A and B show the immunofluorescence staining results of NGF and CNTF in frozen sections on days 3 and 7 post-injury, respectively, for LysM-Cre MCT1. fl / fl The relative fluorescence intensity of NGF in the corneal tissue of the control group mice was significantly lower than that in the control group; (See attached image) Figure 21 CD showed LysM-Cre MCT1 on day 3 post-injury. fl / fl The relative fluorescence intensity of CNTF in the corneal tissue of the control group was significantly lower than that of the control group.

[0068] The above results confirm that macrophage MCT1 can regulate the corneal nerve regeneration process by modulating the local inflammatory microenvironment of the cornea, thereby affecting the expression level of neurotrophic factors (NGF, CNTF).

[0069] (3) Macrophage-specific MCT1 knockout can alter cell metabolism and inflammatory cytokine expression, and inhibit macrophage phagocytic activity in vitro. This invention further investigates the effect of MCT1 deficiency on macrophage phagocytic function. (Appendix) Figure 22 A. Appendix Figure 22 B shows the imaging analysis of macrophages after staining with the macrophage marker CD68 antibody and fluorescent secondary antibody, compared with the control group, LysM-Cre MCT1 fl / fl The percentage of cells engulfing fluorescent microspheres in mouse bone marrow-derived macrophages (BMDMs) was significantly reduced.

[0070] To elucidate its mechanism of action, this study examined the mRNA expression levels of phagocytosis-related genes in two groups of macrophages. (Appendix) Figure 22 C shows the results: the mRNA expression levels of multiple phagocytosis-related surface receptors in macrophages were significantly downregulated in the MCT1 knockout group, including mannose receptor (CD206), complement receptor 3 (CR3), milk fat globule epidermal growth factor 8 (MFG-E8), and macrophage collagen-like structure receptor (MARCO); while the mRNA expression level of class B scavenger receptor 1 (SCARB1) was not significantly different from that in the control group.

[0071] Intracellular metabolic patterns are key factors determining macrophage phenotype and function. Based on this, this invention uses the Seahorse XF Analyzer to detect the glycolytic and oxidative metabolic capacities of mature bone marrow macrophages derived from macrophage-specific MCT1 knockout mice and control mice. Figure 23 A and D illustrate the real-time measurement of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using live-cell experiments. (See attached diagram.) Figure 23 C shows that the basal oxygen consumption and FCCP-induced maximum respiratory oxygen consumption of macrophages in the MCT1 knockout group were significantly reduced, and these indicators are core parameters for evaluating cellular aerobic oxidative metabolism. Notably, compared with the control group, the reserve respiratory capacity of macrophages in the knockout group was significantly decreased, suggesting impaired adaptability to increased energy demand. (Appendix) Figure 23 F shows that the basal extracellular acidification rate of knockout macrophages and the maximum extracellular acidification rate after oligomycin treatment were both significantly increased.

[0072] The above results indicate that MCT1 deficiency in macrophages impairs mitochondrial oxidative phosphorylation and promotes cellular glycolysis. (Appendix) Figure 23 B shows a significant decrease in ATP production from aerobic oxidation in the knockout group of macrophages, suggesting that MCT1 deficiency leads to a reduction in cellular oxidative phosphorylation and metabolic energy supply. (Appendix) Figure 23 G and H show the gene expression detection results. The mRNA expression levels of hexokinase 2 (HK2) and lactate dehydrogenase A (LDHA), key enzymes in glycolysis, were significantly higher in the knockout group than in the control group; while the attached... Figure 23 Images I, J, K, and L show that the mRNA expression levels of key oxidative phosphorylation enzymes—fumarate hydratase (FH), isocitrate dehydrogenase 2 (IDH2), succinate dehydrogenase complex A subunit (SDHA), and pyruvate dehydrogenase complex A1 subunit (PDHA1)—were significantly downregulated. These results further confirm that MCT1 knockout promotes glycolysis in macrophages while inhibiting their aerobic oxidative metabolism.

[0073] In summary, the above experimental results indicate that MCT1-deficient macrophages undergo metabolic reprogramming in vitro, specifically manifested as suppressed oxidative phosphorylation and enhanced glycolysis.

[0074] To further investigate whether MCT1 deficiency promotes the transformation of macrophages to a pro-inflammatory phenotype by regulating macrophage polarization and cytokine secretion, mature bone marrow macrophages from two groups of mice were stimulated for 24 hours with either 1 μg / mL lipopolysaccharide (LPS, which induces M1 polarization) or 100 ng / mL interleukin-4 (IL-4, which induces M2 polarization). Figure 24 A shows the results of real-time quantitative polymerase chain reaction (RT-PCR), which indicate that after LPS stimulation, the mRNA expression level of IL-1β in macrophages of the knockout group was significantly higher than that of the control group; Appendix Figure 24 Figures B and C show that after IL-4 stimulation, the mRNA expression levels of arginase 1 (Arg1) and chitinase-like protein 3 (Ym1) in the knockout group macrophages were significantly lower than those in the control group. There was no significant difference in the basal expression levels of the aforementioned pro-inflammatory and pro-regenerative factors in the macrophages of the two groups.

[0075] The above results suggest that macrophage MCT1 deficiency can promote the expression of macrophage-related pro-inflammatory factors after M1 polarization stimulation, while inhibiting the expression of macrophage-related pro-regenerative factors after M2 planned stimulation, ultimately leading to macrophage polarization imbalance.

[0076] In summary, this study proposes a potential mechanism model for macrophage MCT1 regulation of corneal nerve regeneration. Macrophage MCT1 deficiency first impairs mitochondrial oxidative phosphorylation function, thereby inducing upregulation of pro-inflammatory cytokines (such as IL-1β) and downregulation of regenerative factors (such as Arg1 and Ym1), accompanied by a decrease in the expression level of phagocytic receptors (such as MARCO and MFG-E8). Ultimately, it disrupts the corneal nerve repair microenvironment through macrophage polarization imbalance and impaired phagocytic function, thus hindering the corneal nerve regeneration process.

[0077] (4) MCT1 knockout in macrophages can inhibit axonal growth of trigeminal neurons in vitro. Appendix Figure 25 The following steps are shown for conducting in vitro experiments using the Transwell co-culture system: upper chamber inoculation. Mouse bone marrow-derived macrophages (BMDM) were inoculated into the lower chamber of wild-type (WT) mouse trigeminal neurons. (See attached image) Figure 26 A shows the comparison of axonal branching and extension capabilities of neurons co-cultured with BMDM after 24h and 48h using Sholl analysis and immunofluorescence staining of βⅢ microtubules; Appendix Figure 26AC showed that, according to Sholl analysis, after 24 hours of co-culture, the complexity of branches directly emanating from the cell bodies of neurons in the knockout group was significantly lower than that in the control group. Figure 26 AB shows that the maximum number of branch intersections per neuron in the two groups of neurons is 4.750 ± 1.506. With 8.00±2.385 per neuron The control group showed a significantly higher number of neuronal fiber branches and the number of fibers originating from the cell body compared to the knockout group. (See attached image) Figure 26 C shows that at the higher-order branching level (secondary and higher branches), the number of individual neuron branch intersections at 24h and 48h was significantly reduced in neurons after co-culturing with knockout BMDMs compared to the control group. (Appendix) Figure 26 D shows the measurement of the average axon length of a single neuron. The average fiber length of a single neuron in the knockout group was significantly lower than that in the control group at both 24h and 48h of co-culture. .

[0078] To explore the reasons for the difference in TG cell growth between the two groups, cell culture supernatant was extracted and ELISA was performed. The results showed that... Figure 26 E showed that after 48 hours of co-culture, the concentration of IL-1β protein in the supernatant of the knockout group was significantly higher than that in the control group, while the attached... Figure 26 F showed that the Arg-1 protein concentration was significantly lower than that in the control group, suggesting that MCT1 knockout in macrophages can inhibit the growth of trigeminal neuron axons in vitro by regulating the expression of IL-1β and Arg-1.

[0079] (5) Subconjunctival injection of MCT1-functional macrophages can improve the delayed regeneration of damaged corneal nerves in MCT1-deficient mice. This invention aims to verify whether the inhibitory effect of macrophage MCT1 functional defects on corneal nerve injury repair can be reversed. Figure 28 This demonstrates the macrophage-specific MCT1 knockout mouse (LysM-Cre MCT1). fl / fl Timeline of subconjunctival injection of functional MCT1-derived bone marrow macrophages (BMDMs). (See attached...) Figure 28 A shows the conjunctival condition of mice after subconjunctival injection, performed on day 3 following corneal injury. This time point coincides with the time window for circulating macrophages to infiltrate the injured nerve tissue. The macrophage preparation procedure was as follows: bone marrow-derived macrophages were isolated from wild-type mice 7 days prior to injection, induced to mature in vitro, and then... Figure 29 C shows the use of flow cytometry to identify its cell phenotype.

[0080] Appendix Figure 29A shows the corneal sensitivity test results on days 7 and 14 after corneal epithelial curettage. Subconjunctival injection of macrophages significantly promoted LysM-Cre MCT1. fl / fl The corneal sensitivity of the mice recovered, and their corneal sensitivity level returned to that of the control mice (MCT1) on day 14. fl / fl The degree of similarity.

[0081] Appendix Figure 28 B illustrates corneal nerve regeneration as shown by immunofluorescence staining of βⅢ microtubule protein in mouse corneal tissue at days 7 (D7) and 14 (D14) post-injury. (See attached image.) Figure 29 BC showed the condition on day 7 post-injury, whether in LysM-Cre MCT1 fl / fl Mice or MCT1 fl / fl In mice, there was no significant difference in corneal nerve density and the number of nerve branch points between the eyes that received macrophage injections and those that received phosphate-buffered saline (PBS) injections. However, by day 14 post-injury, LysM-Cre MCT1... fl / fl In mice, the eyes that received macrophage injections had significantly higher corneal nerve density and the number of nerve branch points than the eyes that received PBS injections; conversely, MCT1... fl / fl In mice, the corneal nerve density and the number of nerve branch points in the eyes that received macrophage injections were significantly lower than those in the eyes that received PBS injections.

[0082] MCT1 fl / fl The damaging effect observed in mice on day 14 post-injury may be due to exogenous macrophages aggravating local corneal inflammation, thereby delaying nerve regeneration. In contrast, macrophage injection significantly improved LysM-Cre MCT1. fl / fl The corneal nerve regeneration capacity of the mice was restored to that of MCT1 mice that received PBS injections. fl / fl The levels were similar to those in the control group. This result confirms that local delivery of macrophages with intact MCT1 function can alleviate the delayed corneal nerve regeneration caused by macrophage MCT1 deficiency. This improvement is compensatory—exogenous macrophage injection did not produce a similar beneficial effect in mice with pre-existing macrophage MCT1 function.

[0083] It should be understood that the exemplary embodiments described herein should be considered in a descriptive sense only and not for limiting purposes. The features or aspects described in the various embodiments should generally be considered to be feasible for similar features or aspects in other embodiments as well.

[0084] SEQ ID NO:1: Nucleic acid sequence encoding mouse-derived MCT1 protein carried by the AAV vector

Claims

1. Application of a nucleic acid vector encoding the macrophage monocarboxylic acid transporter MCT1 in the preparation of a pharmaceutical composition for promoting corneal nerve regeneration and recovery of corneal sensitivity after corneal nerve injury.

2. The application according to claim 1, characterized in that: The nucleic acid vector is a macrophage-targeted or macrophage-biased expression vector that can enhance the expression level of MCT1 in macrophages.

3. The application according to claim 1 or 2, characterized in that: The corneal nerve injury includes damage to the structure or function of the corneal nerve caused by mechanical damage to the corneal epithelium, corneal surgery, chemical damage, post-infectious damage, diabetic corneal neuropathy, or neurotrophic corneal disease.

4. The application according to claim 1 or 2, characterized in that: The nucleic acid vector promotes corneal nerve regeneration and corneal sensitivity recovery by increasing macrophage MCT1 levels, enhancing macrophage oxidative phosphorylation levels, reducing glycolysis levels, limiting the expression of IL-1β and / or Lyz1 pro-inflammatory factors, and increasing the levels of Arg1, Ym1, NGF and / or CNTF pro-regeneration or neurotrophic factors.

5. The application according to any one of claims 1 to 4, characterized in that: The nucleic acid vector contains a promoter and a nucleic acid sequence encoding MCT1, wherein the promoter is the macrophage-specific promoter F4 / 80 or a promoter capable of driving expression in macrophages.

6. The application according to claim 5, characterized in that: The nucleic acid vector is an adeno-associated virus vector, preferably an AAV2 / 9 vector.

7. The application according to any one of claims 1 to 6, characterized in that: The nucleic acid sequence encoding MCT1 includes the mouse MCT1 coding sequence shown in SEQ ID NO:

1.

8. The application according to any one of claims 1 to 7, characterized in that: The pharmaceutical composition is a formulation suitable for ophthalmic administration, subconjunctival administration, periorbital administration, or intravenous administration.

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