Application of vitamin A in preparation of medicine for treating visual impairment related to mitochondrial DNA mutation

Abstract

The invention provides application of vitamin A in preparation of a medicine for treating visual impairment related to mitochondrial DNA mutation. The vitamin A comprises derivatives thereof, and the related visual impairment comprises Leber hereditary optic neuropathy, retinitis pigmentosa and other mitochondrial retinopathy. The specific implementation scheme comprises the following steps: adding vitamin A or pharmaceutically acceptable salts and ester derivatives thereof into a daily feed for mice and adding into a cell culture medium. The invention proves that the supplement of vitamin A can recover abnormal mitochondrial function in retinal cells of mutated mice, correct disorder of visual signaling pathways, reduce oxidative stress and improve the forms and functions of retinal ganglion cells, photoreceptor cells and Mueller cells, and the supplement of vitamin A can effectively recover the expression level of mitochondrial genes and reduce oxidative stress.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the use of vitamin A in the preparation of drugs for vision impairment associated with mitochondrial DNA mutations, and is particularly suitable for mitochondrial retinal diseases such as Leber hereditary optic neuropathy and retinitis pigmentosa. Background Art

[0002] There is currently no effective treatment for visual impairment caused by mitochondrial DNA mutations, such as LHON. LHON is a degenerative disease of the optic nerve caused by mitochondrial DNA mutations (e.g., ND6 P25L), characterized by degeneration of retinal ganglion cells, thinning of the retinal nerve fiber layer, and related visual dysfunction. Patients suffer severe bilateral vision loss or even blindness, severely impacting their quality of life. Existing treatments, such as gene therapy and coenzyme Q10, have limited effectiveness and cannot reverse existing cell damage. Studies have found that mtDNA mutations can lead to abnormal mitochondrial function in retinal cells, accumulation of reactive oxygen species (ROS), and damage to the vitamin A metabolic pathway. Vitamin A is a core substance in the visual cycle, but its repair role in mitochondrial retinopathy has not yet been revealed. Therefore, the development of new vitamin A-based therapies has important clinical significance. This application reveals for the first time that vitamin A significantly improves the pathological process of mitochondrial retinopathy by regulating mitochondrial function, resisting oxidative stress, and restoring the visual signaling pathway. Summary of the Invention

[0003] The purpose of the present invention is to provide the use of vitamin A in preparing a medicine for visual impairment associated with mitochondrial DNA mutation, which is a new pharmaceutical use of vitamin A.

[0004] The vitamin A includes its derivatives, which are retinol and / or retinol acetate and its derivatives.

[0005] In the pharmaceutical use provided by the present invention, vitamin A plays a protective role on the retina in mitochondrial DNA mutation-related visual impairment, wherein the mitochondrial DNA mutation-related visual impairment is a mitochondrial retinal disease such as Leber hereditary optic neuropathy (LHON) or retinitis pigmentosa.

[0006] Embodiments of the present invention also provide a drug for treating vision impairment associated with mitochondrial DNA mutations, comprising vitamin A and pharmaceutically acceptable excipients. The drug concentration is 0.1 mg to 10 mg / ml, and the vitamin A dosage is 10,000 to 120,000 IU / kg. Administration is continued for four days. Administered orally.

[0007] During their research into vision impairment associated with mitochondrial DNA mutations, the inventors of this application discovered that mitochondrial DNA mutations trigger abnormal retinal vitamin A metabolism and component deficiencies. Vitamin A and its derivatives play a crucial role in retinal development and visual function, and vitamin A supplementation can correct vision impairment caused by mitochondrial DNA mutations.

[0008] The present invention provides a mouse model capable of detecting the restorative effect of vitamin A on retinal diseases, wherein the mouse model is a C57BL / 6J background mouse with the m.13997G>A mutation.

[0009] The present invention provides a cell line capable of detecting the effect of vitamin A on restoring mitochondrial function. The cell line is preferably a mammalian cell, and more preferably, the mammalian cell is a cytoplasmic fusion cell line with the m.14484T>C mutation. This cell line is modified from 143B cells purchased from the Cell Bank of the Chinese Academy of Sciences (Catalog No. TCHu264).

[0010] The present invention uses a mitochondrial mutation mouse model with a C57BL / 6J background. Starting from 2 months of age, retinol acetate is added to the mouse feed, and the therapeutic effect on retinal morphology and function is evaluated after 2 months of treatment. Patient-derived cell lines are used, retinol is added to the cell culture medium, and the recovery effect of mitochondrial function is analyzed after 4 days of treatment. The above experimental results confirm that vitamin A supplementation can effectively restore the mitochondrial morphology and function in the retinal cells of mutant mice, correct abnormal vitamin A metabolism, and improve retinal structure and function. In patient-derived cells, vitamin A supplementation can effectively restore mitochondrial gene expression levels and reduce reactive oxygen pressure.

[0011] Compared with the prior art, the present invention has the following advantages: 1. Able to effectively restore retinal cell damage and mitochondrial dysfunction caused by mitochondrial gene mutations; 2. It has significant therapeutic effects in improving retinal electrophysiological indicators, restoring the number of retinal ganglia, and adjusting visual signal conduction; 3. The drug administration method is flexible, safe, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 2 is a comparison of optical coherence tomography images of the mouse retina before and after treatment in Example 1.

[0013] Figure 2 This is a diagram showing the results of detecting retinal ganglion cells in mouse retina using immunofluorescence technology in Example 1.

[0014] Figure 3 This is a graph showing the results of detecting the number of neurites in mouse retinal Müller cells using immunofluorescence technology in Example 1.

[0015] Figure 4 This is a graph showing the results of detecting the number of photoreceptor cells in the mouse retina using immunofluorescence technology in Example 1.

[0016] Figure 5 This is a diagram showing the results of detecting the retinol content in the mouse retina using enzyme-linked immunosorbent assay in Example 1.

[0017] Figure 6 This is a graph showing the results of the mouse retinal electrophysiological dark response test in Example 1, showing the changes in b-wave amplitude.

[0018] Figure 7 This is a graph showing the results of the mouse retinal electrophysiological bright response test in Example 1, showing the changes in b-wave amplitude.

[0019] Figure 8 This is a comparison of the morphology of mouse retinal mitochondria detected by transmission electron microscopy in Example 1.

[0020] Figure 9 This is a graph showing the results of detecting the ATP content in mice using luciferase in Example 1.

[0021] Figure 10 This is a graph showing the results of detecting the ATP content in mice using chemiluminescence in Example 2.

[0022] Figure 11 This is the result of detecting the expression level of mitochondrial genes in cells by protein immunoblotting in Example 3.

[0023] Figure 12 This is a graph showing the results of detecting cellular reactive oxygen species levels using flow cytometry in Example 3. DETAILED DESCRIPTION

[0024] In order to make the purpose and beneficial effects of the present invention more clear, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments. However, ordinary technicians in this field can make various deformation and modifications to the present invention without departing from the spirit and technical solutions of the present invention. All these deformation and modifications should be within the scope of protection of the present invention.

[0025] Example 1 Application of 120,000 IU / kg Vitamin A Supplementation in Treating Retinopathy in Mitochondrial Mutation Mice

[0026] 1. Experimental Grouping Wild-type and mitochondrial ND6 P25L mutant mice with a C57BL / 6J background were selected and divided into four groups: wild-type mice fed a control diet, wild-type mice fed a vitamin A-supplemented diet, ND6 mutant mice fed a control diet, and ND6 mutant mice fed a vitamin A-supplemented diet.

[0027] 2. Vitamin A supplementation The control diet contained 4000 IU / kg of vitamin A, while the vitamin A-supplemented diet contained 120,000 IU / kg of vitamin A. The vitamin A in the diet was present as retinyl acetate. Starting at 2 months of age, wild-type and mutant mice were randomly assigned to one of the diet groups. All mice were maintained under a 12:12-hour light / dark cycle for 8 weeks with free access to food and water for longitudinal analysis of fundus phenotypes. This study adhered to ethical guidelines and animal experimentation ethics regulations, and relevant experimental permits and approvals were obtained.

[0028] 3. Treatment outcome evaluation

[0029] (1) Retinal fundus examination and optical coherence tomography (OCT) Figure 1 The following images show the results of continuous retinal fundus examinations and optical coherence tomography (OCT) scans of mice at 2, 3, and 4 months of age. The results show that mutant mice fed the control diet developed gradually expanding retinal fundus lesions, and OCT examinations of the corresponding areas revealed structural abnormalities in rod photoreceptors and the retinal pigment epithelium. However, vitamin A supplementation significantly reduced the retinal fundus lesions in ND6 P25L mutant mice.

[0030] (2) Retinal cell morphology detection Figure 2 、 Figure 3 、 Figure 4 The figures show the number of Brn3a-positive ganglion cells, Müller cell neurites, and photoreceptor cells in the mouse retina detected by retinal immunofluorescence. The results show that vitamin A supplementation significantly restored the number of Brn3a-positive ganglion cells, Müller cell neurites, and photoreceptor cells in the mutant mouse retina.

[0031] (3) Retinal vitamin A content detection Figure 5 The figure shows the results of enzyme-linked immunosorbent assay (ELISA) testing for vitamin A content in the mouse retina. The results show that vitamin A content in the mutant mouse retina increased significantly after vitamin A supplementation, demonstrating the effectiveness of retinal vitamin A supplementation through diet.

[0032] (4) Retinal electrophysiological testing Figure 6 、 Figure 7 The images show the results of full-field retinal electrophysiological dark and light responses in mice. The results show that after vitamin A supplementation, the b-wave amplitude in both dark and light responses of mutant mice was significantly restored, indicating the restoration of photoreceptor visual function.

[0033] (5) Retinal mitochondrial morphology detection Figure 8 Transmission electron microscopy was used to examine the mitochondrial morphology of the ganglion cell layer and inner ganglion layer of the mouse retina. The results showed that vitamin A supplementation restored the cristae structure of the mutant mouse mitochondria.

[0034] (6) Mouse ATP production detection Figure 9 The figure shows the results of luciferase assays for ATP production in mice. The results show that vitamin A supplementation significantly increased ATP production in mutant mice, indicating the restoration of mitochondrial function.

[0035] Example 2 Application of 10000 IU / kg Vitamin A Supplementation in Treating Retinopathy in Mitochondrial Mutation Mice

[0036] 1. Experimental Implementation Wild-type and mitochondrial ND6 P25L mutant mice of a C57BL / 6J background were divided into four groups as described in Example 1. The control group's diet contained 4000 IU / kg of vitamin A, while the vitamin A-supplemented diet contained 10,000 IU / kg of vitamin A. The vitamin A in the diet was present as retinyl acetate. Starting at 3 weeks of age, wild-type and mutant mice were randomly assigned to one of the diet groups. All mice had free access to food and water under a 12:12 hr light / dark cycle for 8 weeks.

[0037] 2. Treatment outcome evaluation

[0038] (1) Detection of ATP production in mice Figure 10 The figure shows the results of chemiluminescence detection of ATP production in mice supplemented with 10,000 IU / kg of vitamin A. The results show that ATP production in mutant mice increased significantly after vitamin A supplementation, indicating the restoration of mitochondrial function.

[0039] Example 3 Application of Vitamin A Supplementation in Treating LHON Patient-Derived Cells

[0040] 1. Experimental Materials Cytoplasmic hybrid cells of LHON patients with mitochondrial ND6 14484T>C mutation and wild-type cells with the same haplotype were selected.

[0041] 2. Vitamin A supplement Vitamin A was added to the cell culture medium of wild-type cells and mutant cells at concentrations of 0.1 μM, 1 μM, and 10 μM, respectively, and the therapeutic effects were evaluated after 4 days of treatment.

[0042] 3. Evaluation of treatment efficacy (1) Analysis of mitochondrial gene expression levels Figure 11 The figure shows the results of Western immunoblotting to detect mitochondrial gene expression levels in cells. The results show that after supplementation with 0.1μM, 1μM, and 10μM vitamin A, the expression levels of ND1, ND4, and ND6 of mitochondrial complex I in mutant cells were significantly restored; (2) Detection of reactive oxygen species levels Figure 12 The figure shows the results of flow cytometry analysis of cellular reactive oxygen species (ROS). The results show that 0.1μM and 1μM vitamin A supplementation reduced the ROS levels in the mutant cells.

[0043] In summary, the present invention provides a novel pharmaceutical composition and administration method based on vitamin A for treating visual dysfunction caused by mitochondrial gene mutations. By regulating the metabolism of vitamin A in the retina and its restorative effect on mitochondrial function, damaged retinal nerve cells can be repaired, thereby improving vision.

Claims

1. Use of vitamin A in the preparation of a drug for mitochondrial DNA mutation-related visual impairment, characterized in that: The vitamin A includes its derivatives, and the related visual impairment is Leber hereditary optic neuropathy or retinitis pigmentosa caused by mitochondrial retinopathy.

2. The use according to claim 1, characterized in that The vitamin A and its derivatives are retinol and / or retinol acetate derivatives.

3. The use according to claim 1, characterized in that The medicine comprises vitamin A and its derivatives and pharmaceutically acceptable excipients.

4. The use according to claim 1, characterized in that The dosage of vitamin A contained in the medicine is 10000-120000 IU / kg.

5. The use according to claim 1, characterized in that The drug concentration is 0.1uM-10uM.

6. The use according to claim 1, characterized in that The route of administration is oral.

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