CN117982516A

CN117982516A - Application of SKQ1 in inhibiting mitochondrial autophagy and preparing product for inhibiting mitochondrial autophagy

Info

Publication number: CN117982516A
Application number: CN202410187102.2A
Authority: CN
Inventors: 雷博; 姚舜; 许瑾; 韩金凤; 杨名珠; 郭庆歌; 李亚
Original assignee: Henan Provincial Eye Hospital
Current assignee: Henan Provincial Eye Hospital
Priority date: 2024-02-20
Filing date: 2024-02-20
Publication date: 2024-05-07

Abstract

The invention provides application of SKQ 1in inhibiting mitochondrial autophagy and preparing a product for inhibiting mitochondrial autophagy, and relates to the technical field of biology. The application comprises any one of the following (I) - (V): preparing a medicament for treating diseases associated with mitochondrial damage, including damage caused by autophagy; (ii) the non-diagnostic and therapeutic destinations inhibit mitochondrial autophagy; (iii) preparing a formulation for inhibiting mitochondrial autophagy; (iv) inhibiting mitochondrial autophagy-related protein expression for non-diagnostic and therapeutic purposes; (V) preparing a preparation for inhibiting the expression of the mitochondrial autophagy-related protein. Wherein SKQ1 can effectively inhibit excessive autophagy of mitochondria and relieve related diseases.

Description

Application of SKQ1 in inhibiting mitochondrial autophagy and preparing product for inhibiting mitochondrial autophagy

Technical Field

The invention relates to the technical field of biology, in particular to application of SKQ1 in inhibiting mitochondrial autophagy and preparing a product for inhibiting mitochondrial autophagy.

Background

There are many factors affecting and impairing mitochondrial function, mainly three aspects, nutrient deficiency, environmental toxins and oxidative damage. Mutations in mitochondrial genes caused by exogenous or endogenous factors can cause some proteins of mitochondria to fail to function normally, which is manifested as respiratory chain dysfunction, reduced ATP production, increased electrons in the mitochondrial matrix, easy combination with oxygen to generate superoxide anions (a form of active oxygen), the increase of active oxygen breaks the steady state of redox reactions, causes depolarization of mitochondrial membrane potential, triggers autophagy of mitochondria, clears damaged mitochondria, and ensures healthy mitochondrial quantity and steady state balance. Excessive autophagy of mitochondria can degrade normal mitochondria, causing apoptosis or death. The eye is more susceptible to damage by mitochondria as metabolically active tissues such as cornea, lens, ciliary body, retina and optic nerve. Typical manifestations of primary mitochondrial disease generally affect: (1) outer retinopathy: retinal pigment degeneration, macular dystrophy, macular degeneration, diabetic retinopathy, retinal degeneration; (2) inner retinopathy: optic atrophy, glaucoma, leber's Hereditary Optic Neuropathy (LHON), autosomal dominant optic neuropathy (ADOA); (3) anterior ocular segment lesions: keratopathy, glaucoma; (4) extraocular myopathy: ptosis, eye paralysis, chronic progressive extraocular muscle paralysis (CPEO); (5) The cerebral cortex and/or white matter, leading to visual field defects and advanced vision processing disorders. In the above diseases, excessive activation of mitochondrial autophagy is an important cause of lesions, however, current drugs against mitochondrial autophagy are not common.

Small molecule drugs targeting mitochondrial autophagy are currently not uncommon. Meanwhile, the existing targeted mitochondrial medicines have unsatisfactory effects, which may be caused by weaker specificity and lower localized concentration to mitochondria, and if the dosage is increased, adverse reactions or side effects may be caused. Therefore, development of new drugs capable of alleviating diseases caused by excessive activation of mitochondrial autophagy is currently demanded in the market.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide application of SKQ1 in inhibiting mitochondrial autophagy and preparing a product for inhibiting mitochondrial autophagy, wherein SKQ1 can effectively inhibit excessive mitochondrial autophagy and relieve related diseases.

In order to solve the technical problems, the invention adopts the following technical scheme:

The invention provides the use of SKQ1 in any one of the following (I) - (V):

Preparing a medicament for treating diseases associated with mitochondrial damage, including damage caused by autophagy;

(ii) the non-diagnostic and therapeutic destinations inhibit mitochondrial autophagy;

(iii) preparing a formulation for inhibiting mitochondrial autophagy;

(iv) inhibiting mitochondrial autophagy-related protein expression for non-diagnostic and therapeutic purposes;

(V) preparing a preparation for inhibiting the expression of the mitochondrial autophagy-related protein.

In alternative embodiments, the mitochondrial damage related disease comprises at least one of an outer retinal damage disease, an inner retinal damage disease, a anterior ocular segment damage disease, an extraocular muscle damage disease, a cerebral cortex lesion, and a leukoencephalopathy;

In alternative embodiments, the outer retinal damage disease comprises at least one of omental pigment degeneration, macular dystrophy, macular degeneration, diabetic retinopathy, and retinal degeneration;

in alternative embodiments, the inner retinal damage disease includes at least one of optic atrophy, glaucoma, leber's hereditary optic neuropathy, and autosomal dominant optic neuropathy;

in alternative embodiments, the ocular anterior segment damaging disease comprises hereditary and non-hereditary keratopathy;

in alternative embodiments, the extraocular muscle damaging condition comprises at least one of ptosis, eye paralysis, and chronic progressive extraocular muscle paralysis;

in alternative embodiments, the cerebral cortical lesions and the leukoencephalopathy include visual field defects and advanced vision processing disorders caused by brain injury.

In alternative embodiments, the inhibiting mitochondrial autophagy in (ii) and (iii) independently comprises inhibiting mitochondrial autophagy in the cell in vitro.

In alternative embodiments, the cells comprise at least one of HEK cells, retinal pigment epithelial cells, and skin fibroblasts;

In alternative embodiments, the skin fibroblasts include autosomal dominant optic neuropathy patient skin fibroblasts and/or leber's hereditary optic neuropathy patient skin fibroblasts.

In alternative embodiments, the inhibiting expression of the mitochondrial autophagy-related protein in (iv) and (v) independently comprises inhibiting expression of the mitochondrial autophagy-related protein in the cell in vitro.

In alternative embodiments, the use comprises contacting the cells with SKQ 1.

In alternative embodiments, the sufficient contact of the cells with SKQ1 comprises treating the cells with SKQ1 at a final concentration of 10-50 nM.

In an alternative embodiment, the sufficient contact of the cells with SKQ1 comprises treating the cells with SKQ1 at a final concentration of 10-50 nM and then treating the cells with SKQ1 at a final concentration of 100-300. Mu.M.

In an alternative embodiment, the mitochondrial autophagy-related proteins of (iv) and (v) each independently comprise at least one of PINK1, BNIP3, and LC 3B.

In an alternative embodiment, the working concentrations of SKQ1 in the formulations in (III) and (V) are each independently 10-50 nM; or 100 to 300 mu M independently.

Compared with the prior art, the invention has the following beneficial effects:

SKQ1 is a lipid quinone derived cation, and the use of SKQ1 to inhibit mitochondrial autophagy has the following advantages: 1) Targeting mitochondria is extremely efficient. SKQ1 has two properties, one is cardiophilic, and cardiolipin is more present in the mitochondrial membrane, so SKQ1 is more prone to bind to mitochondria and less to other organelles in the cell; and secondly, the mitochondria are a special organelle with negative charge, and the inner membrane and the outer membrane of the mitochondria form potential difference, and the potential of the membrane is favorable for attracting SKQ1 to be combined. 2) Recyclability. Under the condition that the mitochondria are seriously damaged and the cytoplasm is in a high oxygen free radical state, the SKQ1 can be oxidized, when the mitochondrial function is improved and the cytoplasm is in a low oxygen free radical state, the SKQ1 can be reduced by an electron transfer chain, and then if the mitochondria are damaged again, the SKQ1 can be recycled, so that the treatment efficiency is greatly improved; 3) The safety is high. The high targeting and recycling property of SKQ1 enables the effective concentration to be lower, and the damage to cells and tissues is extremely small, so that the SKQ1 has higher safety.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the results of a SKQ1 target protein KEGG enrichment assay in example 1;

FIG. 2 shows Western blot detection results of HEK292T cell autophagy-related proteins PINK1 and LC3B and reference protein beta-actin in example 2;

FIG. 3 shows the results of cell viability assays of HEK292T cells treated with SKQ1 and HEK292T cells of the control group in example 2;

FIG. 4 shows Western blot detection results of hTERT-PE1 cell autophagy-related proteins PINK1 and LC3B and an internal reference protein beta-actin in example 2;

FIG. 5 shows the results of cell viability assays of hTERT-PE1 cells treated with SKQ1 and control hTERT-PE1 cells in example 2;

FIG. 6 shows Western blot detection results of the ADOA patient skin fibroblast-associated proteins BNIP3 and LC3B and the internal reference protein beta-actin in example 3;

FIG. 7 shows the results of apoptosis assays of skin fibroblasts from ADOA patients and cells from control group after SKQ1 treatment in example 3;

FIG. 8 shows the Western blot detection results of the skin fibroblast-associated proteins PINK1 and LC3B and the internal reference protein beta-actin of LHON patients in example 4.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

SKQ1 (Visomitin) is a small molecule compound of a methylated derivative of plastoquinone bound to triphenylphosphine cation by decane, and the compound is shown as a formula (I):

SKQ1 can significantly clear mitochondrial ROS and improve mitochondrial microenvironment. SKQ1 is highly specific as a lipophilic cation for it due to the negative charge within the mitochondrial matrix. Under certain conditions, SKQ1 concentrations in the mitochondrial matrix can accumulate 1 x 10 ⁸ times higher than extracellular. The SKQ1 also has extremely strong mitochondrial targeting and little localization to other organelles, so that the effective concentration of the SKQ1 can be as low as nM, the effect is ensured, and the side effect is greatly reduced. Therefore, SKQ1 has the characteristics of high specificity, high efficiency and safety.

Meanwhile, experiments show that SKQ1 is favorable for inhibiting overactivated mitochondrial autophagy so as to maintain mitochondrial homeostasis balance, and achieves the effect of treating diseases. Based on the above, SKQ1 can treat diseases related to mitochondrial injury by inhibiting mitochondrial autophagy, and can play a role in a dual mode by combining the antioxidant activity of the medicine per se, so that the treatment effect is improved.

Based on the above findings, the present invention provides the use of SKQ1 in any one of the following (I) to (V):

And (I) preparing a medicament for treating diseases related to mitochondrial autophagy injury.

In alternative embodiments, the mitochondrial damage related disease comprises at least one of an outer retinal damage disease, an inner retinal damage disease, a anterior segment damage disease, an extraocular muscle damage disease, a cerebral cortex lesion, and a leukoencephalopathy.

In alternative embodiments, the outer retinal damage disease includes, but is not limited to, at least one of omental pigment degeneration, macular dystrophy, macular degeneration, diabetic retinopathy, and retinal degeneration.

In alternative embodiments, the inner retinal damage disease includes, but is not limited to, at least one of optic atrophy, glaucoma, leber's Hereditary Optic Neuropathy (LHON), and autosomal dominant optic neuropathy (ADOA).

In alternative embodiments, the ocular anterior segment damaging disease includes, but is not limited to, hereditary and non-hereditary keratopathy.

In alternative embodiments, the extraocular muscle damaging condition includes, but is not limited to, at least one of ptosis, eye paralysis, and chronic progressive extraocular muscle paralysis (CPEO).

In alternative embodiments, the cerebral cortical lesions and the cerebral leukolesions include, but are not limited to, visual field defects and advanced vision processing disorders caused by brain injury.

In alternative embodiments, the medicament further comprises optional excipients acceptable in the art, including, but not limited to, one or more of solvents, solubilizers, co-solvents, emulsifiers, colorants, fillers, osmotic pressure regulators, stabilizers, glidants, flavoring agents, bacteriostats, suspending agents, fragrances, antioxidants, chelating agents, pH modifiers, adsorbents, surfactants, protectants, humectants, absorbents, diluents, release modifiers, hardening agents, hollow capsules, matrices, and pharmaceutical carrier materials. The medicament may optionally also contain an acceptable amount of residues from the cell culture process, such as culture medium.

In alternative embodiments, the dosage form of the drug may be selected from the group consisting of any pharmaceutically acceptable dosage forms in the art including, but not limited to, injections, inhalants, sprays, oral formulations or drops, such as eye drops. The appropriate dosage form, depending on the route of administration selected, may be made according to common general knowledge in the art, and appropriate excipients are selected.

In alternative embodiments, the subject of the medicament comprises a mammal including, but not limited to, a human, pig, cow, horse, monkey, rat, mouse, guinea pig, sheep, or goat. The subject may be a patient with injury due to mitochondrial autophagy, or may be an animal model of injury due to mitochondrial autophagy.

(II) non-diagnostic and therapeutic targets inhibit mitochondrial autophagy.

In alternative embodiments, the inhibiting mitochondrial autophagy comprises inhibiting mitochondrial autophagy in the cell in vitro.

In alternative embodiments, the cells comprise at least one of HEK cells, retinal pigment epithelial cells, and skin fibroblasts. Wherein the skin fibroblasts are skin fibroblasts of an autosomal dominant optic neuropathy (ADOA) patient and/or skin fibroblasts of a Leber's Hereditary Optic Neuropathy (LHON) patient.

In an alternative embodiment, the mitochondrial autophagy is H ₂O₂ -induced cell-produced mitochondrial autophagy.

In alternative embodiments, inhibiting mitochondrial autophagy in the cell in vitro comprises contacting the cell with SKQ 1.

In alternative embodiments, inhibiting mitochondrial autophagy in cells in vitro comprises treating the cells with SKQ1 at a final concentration of 10 to 50nM (e.g., which may be, but is not limited to, 10, 20, 30, 40, or 50 nM).

In alternative embodiments, inhibiting mitochondrial autophagy in a cell in vitro comprises treating the cell with a final concentration of SKQ1 of 10 to 50nM (e.g., which may be, but is not limited to, 10, 20, 30, 40, or 50 nM), and treating the cell with a final concentration of SKQ1 of 100 to 300 μm (e.g., which may be, but is not limited to, 100, 150, 200, 250, or 300 μm).

In alternative embodiments, the cells are treated with SKQ1 at a final concentration of 10-50 nM for 16-24 h (e.g., which may be, but not limited to, 16, 20 or 24 h).

In alternative embodiments, the cells are treated with SKQ1 at a final concentration of 100-300. Mu.M for 2-6 hours (e.g., which may be, but is not limited to, 2,4, or 6 hours).

In an alternative embodiment, inhibiting mitochondrial autophagy in the cells in vitro comprises treating the cells with SKQ1 at a final concentration of 20nM for 24h.

In an alternative embodiment, inhibiting mitochondrial autophagy in cells in vitro comprises treating cells with SKQ1 at a final concentration of 20nM for 20h and then treating cells with SKQ1 at a final concentration of 200 μM for 4h.

Exemplary non-diagnostic and therapeutic destinations for inhibiting mitochondrial autophagy include, but are not limited to, studying differential expression of related genes or proteins resulting from mitochondrial autophagy, or alterations in physiological and biochemical properties of cells, or developing related agents or drugs that inhibit or promote mitochondrial autophagy, wherein SKQ1 may be used as an agent that regulates the extent of mitochondrial autophagy or as a positive control drug to identify whether the test drug inhibits mitochondrial autophagy.

(III) preparation of preparation for inhibiting mitochondrial autophagy.

In an alternative embodiment, the agent for inhibiting mitochondrial autophagy is used to inhibit mitochondrial autophagy in a cell in vitro.

In an alternative embodiment, the agent for inhibiting mitochondrial autophagy is used to inhibit autophagy in vitro, including at least one of HEK cells, retinal pigment epithelial cells, and skin fibroblasts. Wherein the skin fibroblasts comprise ADOA patient skin fibroblasts and/or LHON skin fibroblasts.

In alternative embodiments, the working concentration of SKQ1 in the agent for inhibiting mitochondrial autophagy is 10-50 nM, such as, but not limited to, 10, 20, 30, 40 or 50nM.

In alternative embodiments, the working concentration of SKQ1 in the formulation for inhibiting mitochondrial autophagy is between 100 and 300. Mu.M, such as but not limited to 100, 150, 200, 250 or 300. Mu.M.

In alternative embodiments, the formulation for inhibiting mitochondrial autophagy further comprises optional excipients acceptable in the art, including, but not limited to, one or more of solvents, pH modifiers, buffer salts, and preservatives.

(IV) non-diagnostic and therapeutic targets inhibit mitochondrial autophagy-related protein expression.

In alternative embodiments, the autophagy-related protein comprises at least one of PINK1, BNIP3, and LC 3B.

In alternative embodiments, the inhibiting expression of the mitochondrial autophagy-related protein comprises inhibiting expression of the mitochondrial autophagy-related protein in the cell in vitro.

In an alternative embodiment, said inhibiting expression of a mitochondrial autophagy-related protein in the cell in vitro comprises inhibiting autophagy in at least one of HEK cells, retinal pigment epithelial cells, and skin fibroblasts in vitro. Wherein the skin fibroblasts comprise ADOA patient skin fibroblasts and/or LHON skin fibroblasts.

In alternative embodiments, inhibiting expression of a mitochondrial autophagy-related protein in a cell in vitro comprises contacting the cell with SKQ 1.

In alternative embodiments, inhibiting expression of a mitochondrial autophagy-related protein in a cell in vitro comprises treating the cell with SKQ1 at a final concentration of 10 to 50nM (which may be, for example, but not limited to, 10, 20, 30, 40, or 50 nM).

In alternative embodiments, inhibiting expression of a mitochondrial autophagy-related protein in a cell in vitro comprises treating the cell with a final concentration of SKQ1 of 10 to 50nM (which may be, for example, but not limited to 10, 20, 30, 40 or 50 nM), and further treating the cell with a final concentration of SKQ1 of 100 to 300 μm (which may be, for example, but not limited to 100, 150, 200, 250 or 300 μm).

In an alternative embodiment, inhibiting expression of a mitochondrial autophagy-related protein in a cell in vitro comprises treating the cell with SKQ1 at a final concentration of 20nM for 24 hours.

In an alternative embodiment, inhibiting expression of a mitochondrial autophagy-related protein in a cell in vitro comprises treating the cell with SKQ1 at a final concentration of 20nM for 20h and treating the cell with SKQ1 at a final concentration of 200 μM for 4h.

Exemplary non-diagnostic and therapeutic destinations for inhibiting mitochondrial autophagy-related protein expression include, but are not limited to, studying differential expression of related genes or proteins resulting from mitochondrial autophagy, or changes in physiological and biochemical properties of cells, or developing agents or drugs that modulate mitochondrial autophagy-related proteins, wherein SKQ1 may serve as a control agent that modulates the extent of mitochondrial autophagy or modulates autophagy-related protein expression, or identifying whether a test drug modulates autophagy-related proteins.

In an alternative embodiment, the agent for inhibiting expression of a mitochondrial autophagy-related protein is used to inhibit expression of an autophagy-related protein including at least one of PINK1, BNIP3, and LC 3B.

In an alternative embodiment, the agent for inhibiting expression of a mitochondrial autophagy-related protein is used to inhibit expression of a mitochondrial autophagy-related protein in a cell in vitro.

In an alternative embodiment, the agent for inhibiting expression of a mitochondrial autophagy-related protein is used to inhibit expression of an autophagy-related protein in at least one of HEK cells, retinal pigment epithelial cells, and skin fibroblasts. Wherein the skin fibroblasts comprise ADOA patient skin fibroblasts and/or LHON skin fibroblasts.

In alternative embodiments, the working concentration of SKQ1 in the formulation for inhibiting expression of the mitochondrial autophagy-related protein is between 10 and 50nM, such as, but not limited to, 10, 20, 30, 40 or 50nM.

In alternative embodiments, the working concentration of SKQ1 in the formulation for inhibiting expression of the mitochondrial autophagy-related protein is between 100 and 300. Mu.M, such as, but not limited to, 100, 150, 200, 250 or 300. Mu.M.

In alternative embodiments, the formulation for inhibiting expression of a mitochondrial autophagy-related protein further comprises an optional acceptable adjuvant in the art including, but not limited to, one or more of a solvent, a pH adjuster, a buffer salt, and a preservative.

The invention is further illustrated by the following specific examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the invention in any way.

Example 1

1. Network pharmacology analysis:

the SKQ1 targeted molecular proteins were retrieved and downloaded in three databases of STITCH (http:// stinch. Embl. De /), SEA (SIMILARITY ENSEMBLE APPROACH, https:// SEA. Bkslab. Org /) and TARGETNET (http:// targetnet. Scbdd. Com /), for a total of 124, as shown in Table 1:

TABLE 1SKQ1 targeting proteins

GPX4	KEAP1	DUX4	MC3R	SIRT3	LCN2
						GUCA1A	APOBEC3A	GPX8	CYBB	BDNF	MCU
SERPINF1	VCAM1	IL1B	SIRT2	CAT	NFKB1
						ANG	CCL2	UCP2	GTF2H1	CYP3A4	AHR
IL6	INS	ERAL1	SOD3	PRDX5	BECN1
						GSHR	S1PR4	MAPK1	MMP9	GREB1	CD4
MAP2K1	PPARGC1A	CES1	NR2F2	MAP3K11	TP53
						PITX1	MAP2K2	SHC1	UCP1	S1PR2	DRD5
MAPK14	EPX	UBD	IGFBP4	GSTO1	ECSIT
						CES2	RIPK2	SOD1	CYP1A2	ANK1	MAPKAPK2
ATG5	TGFB1	TLR9	TNF	ICAM1	CYP1B1
						CNBP	CYP2B6	TXN	ALPL	CD40LG	CDH1
POR	DCTN3	VDAC1	HSD17B6	HDAC4	GSR
						TRIM44	CD8A	TRPM8	FPR1	BCL2L1	DUSP3
GABPA	CKMT1B	CRYAB	TFF1	SYK	CYP1A1
						MGLL	PARK2	NDUFA4	MFN1	ULK1	PINK1
NDUFS4	DHODH

2. KEGG analysis was performed on SKQ1 target proteins, as shown in fig. 1, with the first 5-fold enrichment being the neural degradation pathway (Pathways of neurodegeneration), lipid and atherosclerosis (Lipid and atherosclerosis), reactive oxygen species (Reactive oxygen species), fluid shear stress and atherosclerosis (Fluid SHEAR STRESS AND atherosclerosis) and mitochondrial autophagy (Mitophagy), respectively. It was demonstrated that SKQ1 may play a role in regulating mitochondrial autophagy.

Example 2

SKQ1 inhibits H ₂O₂ -induced mitochondrial autophagy

(1) HEK292T cells were seeded in 6-well plates and pretreated with SKQ1 at a final concentration of 20nM for 20h after cell attachment, control group using DMSO. After 20h pretreatment, the cells were treated with 200. Mu. M H ₂O₂ final concentration for 4h and the control group was basal medium. Mitochondrial autophagy-related proteins PINK1, LC3B and internal reference protein beta-actin were detected by Western blot (FIG. 2). After the same treatment, cell viability was examined using CCK8, and as a result, it was seen that treatment with SKQ1 significantly improved cell viability (fig. 3). It is demonstrated that SKQ1 inhibits autophagy in mitochondria and increases cell viability.

(2) Retinal pigment epithelial cells (a retinal cell) hTERT-PE1 cells were seeded in 6 wells and pretreated with SKQ1 at a final concentration of 20nM for 20h after cell attachment, with DMSO in control. After pretreatment for 20H, the cells were treated with H ₂O₂ at a final concentration of 200. Mu.M for 4H, and basal medium was used for the control group. Mitochondrial autophagy-related proteins PINK1, LC3B and internal reference protein beta-actin were detected by Western blot (FIG. 4). After the same treatment, cell viability was examined using CCK8, and as a result, it was seen that treatment with SKQ1 significantly improved cell viability (fig. 5). It is demonstrated that SKQ1 inhibits overactive mitochondrial autophagy, improves mitochondrial function, promotes cell survival, and thus achieves therapeutic effects in cells of the retinal type.

Example 3

ADOA patients and healthy volunteers were seeded with skin fibroblasts in 6 wells and treated with SKQ1 at a final concentration of 20nM for 24h after cell attachment, and DMSO was used for the control group. Mitochondrial autophagy-related proteins BNIP3, LC3B and internal reference protein beta-actin were detected by Western blot. After the same treatment, apoptosis levels were detected using flow cytometry. The results show that SKQ1 can down-regulate the expression levels of the mitochondrial autophagy-related proteins BNIP3 and LC3B in skin fibroblasts of ADOA patients, indicating that mitochondrial autophagy can be inhibited (fig. 6). Meanwhile, SKQ1 can further inhibit apoptosis (fig. 7). ADOA is a mitochondrial damaging ocular disease, with excessive activation of mitochondrial autophagy. Since the retinal tissue of the patient is not available, we take the skin tissue of the patient for relevant experimental study. Although the skin tissue of the patient is not ocular cells, the skin tissue has special advantages compared with engineering cells such as HKE293T and hTERT-RE1, the genetic background of the tissue cells is consistent with that of the patient, and the experimental result is more in line with clinical practice. Experimental results show that SKQ1 can also inhibit autophagy and apoptosis of mitochondria, promote cell survival and achieve the aim of treating ADOA diseases.

Example 4

Skin fibroblasts from 2 LHON patients and healthy volunteers were seeded in 6 wells and treated with SKQ1 at a final concentration of 20nM for 24h after cell attachment, and DMSO was used for the control group. Mitochondrial autophagy related proteins PINK1 and LC3B and internal reference protein beta-actin are detected by Western blot. The results show that SKQ1 can down-regulate the expression levels of the mitochondrial autophagy-related proteins PINK1 and LC3B in skin fibroblasts of LHON patients, indicating that mitochondrial autophagy can be inhibited (fig. 8). LHON is also a mitochondrial damaging ocular disease, and excessive activation of mitochondrial autophagy.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

Use of skq1 in any one of the following (i) - (v):

Preparing a medicament for treating diseases associated with mitochondrial damage, including damage caused by autophagy;

(ii) the non-diagnostic and therapeutic destinations inhibit mitochondrial autophagy;

(iii) preparing a formulation for inhibiting mitochondrial autophagy;

(iv) inhibiting mitochondrial autophagy-related protein expression for non-diagnostic and therapeutic purposes;

(V) preparing a preparation for inhibiting the expression of the mitochondrial autophagy-related protein.
2. The use according to claim 1, wherein the mitochondrial damage related disease comprises at least one of an outer retinal injury disease, an inner retinal injury disease, an anterior ocular segment injury disease, an outer ocular muscle injury disease, a cerebral cortex lesion, and a leukoencephalopathy;

Preferably, the outer retinal injury disease comprises at least one of omental pigment degeneration, macular dystrophy, macular degeneration, diabetic retinopathy, and retinal degeneration;

Preferably, the inner retinal damage disease includes at least one of optic atrophy, glaucoma, leber's hereditary optic neuropathy, and autosomal dominant optic neuropathy;

preferably, the ocular anterior segment damaging disease includes hereditary and non-hereditary keratopathy;

Preferably, the extraocular muscle injury disease comprises at least one of ptosis, eye paralysis, and chronic progressive extraocular muscle paralysis;

preferably, the cerebral cortex lesions and the leukoencephalopathy include visual field defects and advanced vision processing disorders caused by brain injury.
3. The use of claim 1, wherein said inhibiting mitochondrial autophagy in (ii) and (iii) independently comprises inhibiting mitochondrial autophagy in a cell in vitro.
4. The use according to claim 3, wherein the cells comprise at least one of HEK cells, retinal pigment epithelial cells and skin fibroblasts;

preferably, the skin fibroblasts include skin fibroblasts from patients with autosomal dominant optic neuropathy and/or skin fibroblasts from patients with leber's hereditary optic neuropathy.
5. The use according to claim 1, wherein said inhibiting expression of the mitochondrial autophagy-related protein in (iv) and (v) independently comprises inhibiting expression of the mitochondrial autophagy-related protein in the cell in vitro.
6. The use of claim 5, wherein the cells comprise at least one of HEK cells, retinal pigment epithelial cells, and skin fibroblasts;

preferably, the skin fibroblasts include skin fibroblasts from patients with autosomal dominant optic neuropathy and/or skin fibroblasts from patients with leber's hereditary optic neuropathy.
7. The use according to any one of claims 3 to 6, comprising contacting the cells substantially with SKQ 1.
8. The use of claim 7, wherein the sufficient contact of the cells with SKQ1 comprises treating the cells with SKQ1 at a final concentration of 10 to 50 nM;

Preferably, the cells are treated with SKQ1 at a final concentration of 10-50 nM, and then with SKQ1 at a final concentration of 100-300. Mu.M.
9. The use according to claim 1, wherein the mitochondrial autophagy-related proteins of (iv) and (v) independently comprise at least one of PINK1, BNIP3 and LC3B, respectively.
10. The use according to claim 1, wherein in the formulations of (iii) and (v), the working concentration of SKQ1 is independently 10 to 50nM; or 100 to 300 mu M independently.