WO2015147411A1

WO2015147411A1 - Pharmaceutical composition for prophylaxis or treatment of proliferative vitreoretinopathy containing mitochondrial complex i inhibitor as active ingredient

Info

Publication number: WO2015147411A1
Application number: PCT/KR2014/011970
Authority: WO
Inventors: 유영현; 노지현; 안희배; 정선용
Original assignee: 동아대학교 산학협력단
Priority date: 2014-03-28
Filing date: 2014-12-05
Publication date: 2015-10-01
Also published as: KR101587483B1; KR20150112542A

Abstract

The present invention relates to a pharmaceutical composition for prophylaxis or treatment of proliferative vitreoretinopathy (PVR) containing an intracellular mitochondrial complex I inhibitor as an active ingredient. According to the present invention, the active ingredient which is included in the pharmaceutical composition of the present invention can be developed as an effective therapeutic agent for PVR, an ophthalmic pathological condition, by controlling the cell death of RPE cells. DRAWIMG: FIG. 1: AA % of viability BB TUNEL-positive cell (%) CC + Rotenone DD + siRNA-aB-crystalllin EE Annexin V

Description

Pharmaceutical composition for preventing or treating proliferative vitreoretinopathy containing mitochondrial complex I inhibitor as an active ingredient

The present invention relates to a pharmaceutical composition for preventing or treating proliferative vitreoretinopathy (PVR) containing an inhibitor of intracellular mitochondrial complex I as an active ingredient.

Cell death is a complementary and antagonistic process for cell division, in order to maintain tissue homeostasis, and plays a pivotal role in some physiological processes and diseases. Apoptosis, the most widely studied category, is characterized by mass activation of caspases, staining of contaminated yarn, and reduction of cell volume. Necrosis is characterized by an increase in cell volume, expansion of organelles, and rupture of the plasma membrane, which is usually considered an accidental, uncontrolled type of cell death. Controlled necroptosis is controlled necrotic cell death, which is triggered by extensive caspase inhibition in the presence of death receptor ligands and is characterized by necrotic cell death morphology. Autophagy is a degradable lysosomal pathway, characterized by the accumulation of cytoplasmic material in vacuoles for bulk degradation by lysosomal enzymes. Although autodigestion plays a central role in cell survival, increased autodigestion activity is often associated with cell death. Mitotic catastrophe (MC) is a type of cell death that results from failure of mitotic progression after DNA damage, resulting in tetraploid or endopolyploidy. Cells experiencing MC generally form large cells with multiple micronuclei.

Retinal pigment epithelial (RPE) cells form a single layer of cells adjacent to the photoreceptor outer segment (POS) of the retina, and these cells play a central role in the maintenance of POS cells. RPE cell death is an important factor in some ocular pathological diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). AMD is a progressive degeneration of the macula and is largely classified as dry or wet. The dry form of AMD is more common and is characterized by the presence of drusen in the macula. Mitochondrial DNA variants of Respiratory Complex I are associated with increased AMD risk. Since damage in RPE and its killing is crucial and probably triggers AMD, protection against RPE cell death can delay the onset of AMD. Conversely, RPE cells significantly contribute to the formation of the retinal epithelium in PVR. Thus, the introduction of RPE cell death in the epiretinal membrane may be a new attempt to inhibit cell proliferation in PVR. Most studies of RPE cell death in the context of these ophthalmic pathological conditions have focused on two types of cell death, apoptosis and cell death.

Although advances have been made in understanding RPE cell death, little is known about the role of autodigestion in RPE cell death associated with these ocular pathological conditions. Every day, RPE cells macrophages and digests the distal portion of POS, which ultimately degrades in lysosomes. The interaction of macrophages and autodigestion in RPE requires both POS degradation and maintenance of retinoid levels to support vision. In presbyopic RPE cells, these physiological lysosomal loads can be further increased to further increase the removal of damaged material, and insufficient digestion by old RPE cells of damaged macromolecules and organelles may result in lipofucin ( will cause a gradual accumulation of biological "waste", such as lipofuscin). Thus, abnormalities in lysosomal dependent degradation of exfoliated POS fragments may contribute to the degradation of RPE cells. It has been suggested in previous studies that age-related changes in autodigestion may be based on the genetic sensitivity found in AMD patients and may be related to the pathology of AMD.

However, the mechanism by which autodigestion regulates RPE cell death in AMD is still opaque. The role of autodigestion in the proliferation of RPE cells in PVR and its regulation as a therapeutic strategy for PVR have not yet been documented.

Rotenone is a natural isoflavonoid produced by plants and is a selective and stoichiometric inhibitor of mitochondrial complex I. More specifically, rotenone blocks NADH oxidation by the NADH-ubiquinone oxide reducing agent enzyme complex, resulting in inhibition of mitochondrial respiration and a decrease in ATP synthesis. Rotenone treatment also results in the production of reactive oxygen species (ROS), resulting in cell death. Some studies have shown that rotenone presumably causes accumulation of self-extinguishing vacuoles in response to inhibition of mitochondrial function and production of oxidative stress. Regardless, the activity of rotenone has been studied in various cells, and the effect of rotenone on RPE cells has been little studied. Previous studies using in vitro systems have shown that low concentrations of rotenone result in mtDNA damage in RPE cells, and that increased autodigestion induced by rotenone treatment in older RPE cells results in drusen and It has been suggested that this may affect the formation of AMD. However, the mechanism by which rotenone regulates RPE cell death remains unclear.

The problem to be solved by the present invention is to provide a pharmaceutical composition that can control the cell death of RPE cells for the prevention or treatment of PVR, which is an ophthalmic pathological disease.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of proliferative vitreoretinopathy (PVR) containing a mitochondrial complex I (Mitochodrial complex I) inhibitor as an active ingredient. .

The mitochondrial complex I inhibitor is preferably rotenone.

The active ingredient preferably further comprises an autophagy inhibitor.

The self-extinguishing inhibitor is preferably selected from the group consisting of 3-methyladenine, bafilomycin A1 and chloroquine.

It is preferable that the active ingredient regulates cell death of RPE (Retinal pigment epithelial) cells. The cell death regulation preferably induces apoptosis.

According to the present invention, the active ingredient included in the pharmaceutical composition of the present invention has the potential to be developed as an effective therapeutic agent for the ophthalmic pathological disease PVR by regulating cell death of RPE cells.

1 is a result showing that rotenone induces mitotic catastrophe (MC) in ARPE-19 retinal pigment epithelial cells.

2 shows the results showing that increased autodigestion occurs in RPE-MC cells. Here, C is control, 3MA is 3-methyladenine, Baf is bafilomycin A and CQ is chloroquine.

3 shows results showing that RPE-MC cells are susceptible to inhibition of autophagy.

Figure 4 shows the results showing the increase of RPE-MC cell death by autodigestion inhibition.

Figure 5 shows the results show that Parkin-mediated mitophagy occurs in RPE-CM cells.

6 are the results showing that Mitophagy contributes to the cellular protection of RPE-MC cells.

7 shows the results that alpha B crystallins protect RPE-CM cells from apoptosis.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

We conducted this study to clarify the mechanism that regulates the death of damaged RPE cells by mitochondrial complex I inhibition. As a result, the inventors of the present invention confirmed that rotenone induced MC in RPE cells. In addition, the inventors have identified that RPE cells undergoing mitotic destruction (RPE-MC cells) induced by mitochondrial complex I are susceptible to inhibition of autophagy.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating proliferative vitreoretinopathy (PVR) containing a mitochondrial complex I inhibitor as an active ingredient.

The mitochondrial complex I inhibitor is preferably rotenone.

The rotenone is an isoflavonoid compound produced by plants and acts as an inhibitor of mitochondrial complex I.

The active ingredient preferably further comprises an autophagy inhibitor.

It is preferable that the active ingredient regulates cell death of RPE (Retinal pigment epithelial) cells.

As used herein, "cell death" refers to controlled or uncontrolled death of cells, for example, apoptosis, necrosis, and controlled necroptosis. ), Autophagy and Mitotic catastrophe.

The cell death regulation preferably induces apoptosis.

The apoptosis of RPE cells is promoted by the induction of apoptosis, and the active ingredient of the present invention can be applied as a pharmaceutical composition for preventing or treating proliferative vitreoretinopathy (PVR), which is an ocular pathological disease. .

The pharmaceutical compositions of the present invention may be formulated in various forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, injections of sterile injectable solutions, etc. It can be used orally and can be administered by various routes including intravenous, intraperitoneal, subcutaneous, rectal, topical administration and the like.

Such pharmaceutical compositions may further include carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil Etc. can be mentioned. In addition, the pharmaceutical composition of the present invention may further include a filler, an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.

In a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations comprise at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like are mixed and formulated. In addition, lubricants such as magnesium stearate, talc and the like may also be used in addition to simple excipients.

As a preferred embodiment, oral liquid preparations may be exemplified by suspensions, solvents, emulsions, syrups, and the like, and various excipients, for example, wetting agents, sweeteners, Fragrances, preservatives and the like.

As a preferred embodiment, the preparation for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories and the like. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

The pharmaceutical composition containing the active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, activity of the drug, Sensitivity to drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

In a preferred embodiment, the effective amount may vary depending on the age, sex and weight of the patient, generally between 1 and 10 mg, preferably 2 and 5 mg per kg body weight daily or every other day or divided into 1 to 3 times daily can do. However, the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

The mitochondrial complex I inhibitor is preferably rotenone.

The active ingredient preferably further comprises an autophagy inhibitor.

The cell death regulation preferably induces apoptosis.

Hereinafter, the present invention will be described in more detail with reference to Examples.

<실시예><Example>

1. 재료 및 방법1. Materials and Methods

1.1. 시약1.1. reagent

The following reagents were obtained commercially. Rabbit polyclonal anti-human Bcl-2, Tom20, GFP, YFP, p-Parkin, and mouse monoclonal anti-human Parkin and Beclin-1 antibodies (manufactured by Santa Cruz Biotechnology), rabbit polyclonal anti-human caspas -3, caspase-7, LC3B, HRP-conjugated donkey anti-rabbit, sheep anti-mouse IgG antibody, and RIPA buffer (manufactured by Cell Signaling), rabbit polyclonal anti-human PINK1 (manufactured by Abcam Cambridge), FITC-conjugated goat anti-rabbit and Texas Red-conjugated horse anti-mouse IgGs (manufactured by Vector), Dulbecco's Modified Eagle Medium (DMEM) / F12, Opti-MEM, and penicillin-streptomycin (Manufactured by Gibco BRL), LysoTracker and Lipofectamine (manufactured by Invitrogen), β-actin antibody, Hoechst 33342, dimethyl sulfoxide (DMSO), RNase A, proteinase K, propidium iodide (PI), protein-A Agarose, 3-methyladenine (3MA), Baphylomycin A1 (Baf-A1), Annexin V-FITC Suicide Detection Kit, Mdivi-1, Acridine Orene , Chloroquine, and rotenone (from Sigma), pan caspase inhibitor (from Calbiochem), 5,5 ', 6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimi Dazole Carbocyanine Iodide (JC-1) from Molecular Probes, SuperSignal WestPico Enhanced Chemiluminescent Western Blotting Detection Reagent and Fetal Bovine Serum (FBS) (Thermo), and siPORT Amine (manufactured by Ambion).

1.2. 세포 배양1.2. Cell culture

ARPE-19 cells were purchased from the American Type Culture Collection (ATCC) and maintained at 37 ° C., 5% CO ₂ under an air atmosphere. Cells were grown in a 1: 1 mixture of Ham F12 medium supplemented with DMEM and 10% FBS.

1.3. 로테논 처리1.3. Rotenone treatment

After 24 hours of passage of ARPE-19 cells, the original medium was removed. Cells were washed with PBS and then incubated in the same fresh medium. Rotenone was added to the medium in a stock solution to obtain a 2.5 μM dilution of the drug. After 0, 24, 48, 72, or 96 hours, cells were harvested, stained with trypan blue and counted using a hemocytometer. The concentration of PBS used in the present invention had no effect on ARPE-19 cell proliferation in our previous studies.

1.4. 김사(giemsa) 염색1.4. Gimsa dyeing

ARPE-19 cells were incubated with or without rotenone and then fixed for 10 minutes with methanol. For nuclear morphological observation, slides were stained with Kimsa solution for 15 minutes.

1.5. DNA 저배수성의 정량 및 유세포 분석에 의한 세포 사이클 상 분석1.5. Cell cycle phase analysis by quantification and flow cytometry of DNA low ploidy

Ice-cold 95% ethanol supplemented with 0.5% Tween-20 was added to the cell suspension to a final concentration of 70% ethanol. Fixed cells were pelleted and washed in 1% BSA-PBS solution. Cells were resuspended in 1 mL PBS containing 11 Kunitz U / ml RNase and incubated at 4 ° C. for 30 minutes, washed once with BSA-PBS and resuspended in 50 μg / ml PI solution. Cells were incubated for 30 min at 4 ° C. in the dark, then filtered through a 35 mm mesh and DNA content was determined within 1 hour of staining using a FACSCalibur (Becton-Dickinson) flow cytometer. Cellular DNA content was analyzed using CellQuest software (Becton-Dickinson).

1.6. 공-면역침전1.6. Co-immunoprecipitation

Cell extracts were incubated overnight at 4 ° C. with appropriate antibodies in the extraction buffer. The immunocomplexes were precipitated for 2 hours using Protein A-Sepharose beads and washed 5 times with extraction buffer before boiling in SDS sample buffer. Immunoprecipitated proteins or aliquots containing 40 μg of protein were separated on SDS-polyacrylamide gels and performed as described for Western blot analysis. Each co-immunoprecipitation experiment was confirmed through reciprocal immunoprecipitation.

1.7. 인간 파킨 및 PINK1 siRNA1.7. Human Parkin and PINK1 siRNA

Human Parkin siRNA (SMART pool; L-003603-00-0005) and PINK1 siRNA (SMART pool; L-004030-00-0020) were purchased from Thermo Scientific. As negative controls, the same nucleotides were scraped to form non-genomic combinations.

1.8. siRNA 형질전환1.8. siRNA transformation

siRNA transformation was performed using siPORT amine and Opti-MEM medium. Cells were grown in 6-well plates until reaching 40-50% confluence and transfected to a final siRNA concentration of 100 nM per well. Transformation mixture was added to each well and cells were incubated for 4 hours. Then 2 ml of growth medium was added and the cells were incubated for another 20 hours. After siRNA transformation, the medium was removed and each well was washed in PBS solution.

1.9. 하위세포성 분획화1.9. Subcellular Fractionation

ARPE-19 cells (10 ⁷ cells / well) were washed in Tris-based Mg ²⁺ / Ca ²⁺ -deficient buffer (135 mM NaCl, 5 mM KCl, and 25 mM Tris, pH 7.6) and ice-cold storage ( hypotonic) allowed to swell for 10 minutes in CaRSB buffer (10 mM NaCl, 1.5 mM CaCl ₂ , 10 mM Tris, pH 7.5, and 1 × protease inhibitor cocktail). Cells were ground by 60 strokes in a Dounce homogenizer and stabilized mitochondria by addition of mitochondrial stabilization buffer (210 mM mannitol, 70 mM sucrose, 5 mM EDTA, and 5 mM Tris, pH 7.6). Cells were centrifuged twice at 3,000 rpm for 15 minutes to collect nuclei, and then the supernatant was spun at 14,000 rpm for 20 minutes at 4 ° C. Pele and supernatant contained mitochondria and cytoplasmic fractions, respectively. Cells (5 × 10 ⁷ ) were swollen in ice-cold hypotonic CaRSB buffer and ground using a Dounce homogenizer. The cell homogenate was then allowed to sink by spinning twice at 3,000 rpm for 15 minutes.

1.10. 웨스턴 블롯 분석1.10. Western blot analysis

Cells were washed twice with ice-cold PBS and ice-cold solubilization buffer (300 mM NaCl, 50 mM Tris-HCl [pH 7.6], 0.5% Triton X-100, 2 mM PMSF, 2 μL / mL aprotinin) , And 2 μl / mL leupetin) were resuspended and incubated at 4 ° C. for 30 minutes. The lysates were centrifuged at 14,000 rpm for 15 minutes at 4 ° C. Protein concentrations of cell lysates were determined using the Bradford Protein Assay Kit (Bio-Rad), and equivalent amounts of proteins were determined between 7.5% and 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS). PAGE) was loaded onto the gel. The protein was then transferred onto a nitrogen cellulose membrane (manufactured by Amersham Pharmacia Biotech) and probed with each antibody. Using an enhanced chemiluminescent substrate (SuperSignal West Pico; manufactured by Pierce), immunostaining with antibodies was performed and detected using a photographic film (LAS-3000 Plus; Fuji). Equivalent protein load was confirmed by Ponceau S staining.

1.11. LC3-GFP, YFP-파킨, 및 PINK1-GFP의 ARPE-19 세포로의 형질감염 및 공초점 현미경분석1.11. Transfection and Confocal Microscopy of LC3-GFP, YFP-Parkin, and PINK1-GFP into ARPE-19 Cells

Mammalian expression constructs of human LC3 cloned into pEGFP are: N. Mizushima (Tokyo Medical and Dental University, Tokyo, Japan). YFP hparkin and GFP hPINK1 Provided by J Chung (Seoul National University, Seoul, Korea). ARPE-19 cells were seeded in 6-well plates (10 ⁵ cells / well) and transiently transformed with each construct using Lipofectamine 2000 according to the manufacturer's instructions. Briefly, 2 μg of plasmid DNA was mixed with 6 μl of Lipofectamine 2000 and incubated with Opti-MEM. Plasmid DNA-Lipofectamine 2000 complex was added to the cells and the mixture was further incubated at 37 ° C. for 4 hours. After incubation, the medium was replaced with 2 μl of growth medium and the cells were maintained for an additional 20 hours. Twenty four hours after transformation, plasmid DNA transfection medium was removed and each well was washed with PBS solution. Cells were treated as shown and fixed using 4% paraformaldehyde at 4 ° C. for 15 minutes. Fluorescence images were obtained and analyzed using a Zeiss LSM 510 laser-scanning confocal microscope (Goettingen, Germany). To quantify the cells that exhibited a punctuate pattern, at least 300 cells from each experiment were counted by an observer unknown to the experimental group.

1.12. 투과 전자 현미경1.12. Transmission electron microscope

48 hours after treatment, cells were harvested, pelleted and fixed in 2.5% glutaraldehyde in phosphate buffer. Cells were rinsed with phosphate buffer, samples were post-fixed in 1% osmium tetraoxide for 1 hour, dehydrated in a graded series of ethanol, incubated with propylene oxide and stored overnight at Epon812. Samples were embedded in Epon812 and cured in a 60 ° C. oven. Ultra-thin fragments were obtained using a Reichert Ultracut E microtome. Fragments were stained with uranyl acetate, lead citrate and observed using a transmission electron microscope (Hitachi). For each treatment group or control, at least 200 cells were observed from any selected transmission electron microscope field.

1.13. TUNEL 염색1.13. TUNEL dyeing

Fluorescent TUNEL (terminal deozyribonucleotidyl deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling) analysis to observe cells undergoing apoptosis Was performed using an in situ death detection kit. Briefly, cells grown on coverslips were fixed in 4% paraformaldehyde for 15 minutes and then rinsed in PBS. Cells were incubated with terminal deoxynucleotidyl transferase (TdT) for 1 hour at 37 ° C., and then anti-digoxigenin-FITC was applied for 30 minutes at room temperature. Nuclei were counterstained with propidium iodide / antifade. Fluorescence images were observed and analyzed using a Zeiss LSM 510 laser-scanning confocal microscope. The total cell number of at least 300 cells from each experiment was counted and the number of cells with positive staining was calculated by an observer blinded under epifluorescence optical lens.

1.14. 훼스트(Hoechst) 염색1.14. Hoechst Dyeing

The cells were harvested and the cell suspension was centrifuged on a clear, fat free glass slide using a cell centrifuge. Samples were stained at 37 ° C. for 30 minutes using 4 μg / ml Hoechst 33342 and fixed in 4% paraformaldehyde for 10 minutes. The total cell number from each experiment, which is at least 300 cells, was counted under a DIC optical lens, and the number of cells representing nuclei condensed or fragmented in pest staining was observed under epifluorescent optical lens, with no information on this. Calculated by

1.15. 세포자살, 세포소기관 함량, 및 미토콘드리아 막 전위의 유세포적 측정1.15. Flow cytometry of apoptosis, organelle content, and mitochondrial membrane potential

For quantification of acidic organelle content, cells were incubated for 30 minutes at 37 ° C. in 50 nM LysoTracker red or 1 μg / ml of acridine orange. For quantification of the apoptotic cells, cells were incubated with 500 μl Annexin V-binding buffer comprising 5 μl Annexin V-FITC conjugate and 10 μl propidinium iodide solution. After incubation for 10 minutes at room temperature, cells were analyzed by flow cytometry. For the measurement of mitochondrial membrane potential, cells were incubated with 4 μg / ml JC-1 at 37 ° C. for 30 minutes. Data was acquired and analyzed by ADC XL (Beckman Coulter).

1.16. 통계 분석1.16. Statistical analysis

At least three independent experiments were performed in vitro. The results are expressed as mean ± S.D. From three experiments. The results of the experiments and controls were tested for statistical significance using the Student's t test. In all cases, p values less than 0.05 were considered significant.

2. 결과2. Results

2.1. 로테논은 ARPE-19 망막 색소 상피 세포에서 MC를 유도한다.2.1. Rotenone induces MC in ARPE-19 retinal pigment epithelial cells.

24 hours of rotenone treatment did not reduce the viability of ARPE-19 cells, but 72 hours of treatment significantly reduced cell viability in a dose-dependent manner. Rotenone treatment of 2.5-25 μM for 48 hours slightly reduced (approximately 10-20%) viability of ARPE-19 cells (FIG. 1A). Importantly, most of the ARPE-19 treated with 2.5-25 μM rotenone had a number of nuclei and heterogeneous morphology (FIG. 1B). Since 2.5 μM is the lowest dose that resulted in a slight decrease in the viability of treated ARPE-19 cells for 48 hours, the correlation between rotenone-induced cytotoxicity and cell death and rotenone-induced multinucleation This concentration was used in further studies of the relationship. Flow cytometry analysis showed that most of the rotenone-treated ARPE-19 cells became tetraploid (4C) by 48 hours (FIG. 1C). Since most of the cells involved were> 4N levels of DNA and multinucleated, it seemed possible that these cells could be susceptible to death through MC. Thus, the inventors observed the cells under transmission electron microscopy. Transmission electron microscopy demonstrated the presence of micronuclei in rotenone-treated cells (FIG. 1D). These data show that rotenone induces MC in ARPE-19 cells.

2.2. 증가된 자가소화는 ARPE-19 세포에서 MC를 수반한다.2.2. Increased autodigestion involves MC in ARPE-19 cells.

We next tested whether rotenone increased autodigestion in RPE-MC cells. Western blot analysis demonstrated an increase in LC3-II levels in rotenone-treated ARPE-19 cells (FIG. 2A). Rotenone treatment did not alter the expression level of Beclin-1 or ATG5. However, rotenone slightly increased the expression level of VPS-34 in ARPE-19 cells (FIG. A). Rotenone reduced the interaction between Beclin-1 and Bcl-2, but increased the interaction between Beclin-1 and Vps-34 (FIG. 2B). Flow cytometry was used to observe and quantify cells including organelles labeled LysoTracker or Acridine Orange, which demonstrated that rotenone increased the development of acid vesicle organelles (FIG. 2C). Transmission electron microscopy showed that rotenone increased the number of autophagosomes in RPE-MC cells (FIG. 2D). These data indicate that increased autodigestion is present in RPE-MC cells.

2.3. RPE-MC 세포들은 자가소화 저해에 취약하다.2.3. RPE-MC cells are susceptible to inhibition of autophagy.

The inventors then examined whether inhibition of autodigestion affects the viability of RPE-MC cells. In particular, autophagy inhibitors such as 3MA, bafilomycin A1 (Baf-A1) and chloroquine (CQ) significantly reduced their viability (FIG. 3A). ARPE-19 cells were then transfected with a green fluorescent protein (GFP) -LC3 fusion plasmid, treated with rotenone in the presence or absence of Baf-A1 or chloroquine and then observed using confocal microscopy. After Baf-A1 or chloroquine treatment, the number of LC3-GFP tear points increased significantly (FIG. 3B). Western blot analysis showed that 3MA prevented the accumulation of LC3-II in rotenone-treated ARPE-19 cells. Based on the evaluation of the turnover of LC3-II by Western blot analysis, autodigestion flux analysis showed that the prevention of lysosomal degradation of Baf-A1 or chloroquine led to the accumulation of LC3-II. Rotenone-only treatment induced caspase-3 and caspase-7 cleavage products. Rotenone associated with autodigestion inhibition also induced caspase-3 and caspase-7 incision products. However, inhibition of autodigestion did not increase the accumulation of these incision products compared to rotenone-only treatment (FIG. 3C), indicating that inhibition of autodigestion did not enhance apoptosis. These data indicate that inhibition of autodigestion flux induces death of RPE-MC cells and autodigestion induced by exposing ARPE-19 cells to rotenone is cytoprotective. In addition, we examined whether autodigestion inhibitors affect rotenone-induced multinucleation. Our data show that autodigestion inhibitors did not reverse rotenone-induced multinucleation in ARPE-19 cells (FIG. 3D).

2.4. 자가소화 저해에 의한 RPE-MC 세포 사멸의 증대가 비세포자살성 세포 사멸 경로를 통하여 일어난다. 2.4. Increasing RPE-MC cell death by inhibiting autophagy occurs through a non-apoptotic cell death pathway.

We conducted several analyzes to determine the mechanism of cell death. Western blot analysis demonstrated that caspase-3 and caspase-7 were activated in rotenone alone-treated ARPE-19 cells (FIG. 4A). Flow cytometry demonstrated that the percentage value of Annexin V-positive cells was about 10% (FIG. 4B). In addition, incubation with zVAD-fmk completely prevented cell death induced by rotenone-only treatment. Incubation with zVAD-fmk, however, only partially prevented the increase of cell death by autophagy inhibitors (FIG. 4C). TUNEL analysis demonstrates that the percentage value of TUNEL-positive cells did not increase (FIG. 4D). These data indicate that, with respect to FIG. 3C, autodigestion inhibition mainly induces non-apoptotic cell death in RPE cells undergoing rotenone-induced MC, while rotenone-only treatment in cells in RPE cells. Induces suicide cell death.

2.5. 파킨-매개된 마이토파지(parkin-mediated mitophagy)가 RPE-MC 세포에서 일어난다. 2.5. Parkin-mediated mitophagy occurs in RPE-MC cells.

We then examined whether mitophagy occurred in RPE-MC cells. Western blot analysis showed that rotenone significantly upregulated Parkin protein. Parkin was not in the mitochondria of control cells, but was translocated into the mitochondria of rotenone-treated cells (FIG. 5A). Rotenone also increased the amount of phosphorylated Parkin (p-Parkin) in mitochondria. Rotenone did not significantly upregulate PINK1, and rotenone treatment increased the amount of PINK1 in the mitochondrial fraction. Flow cytometry showed a decrease in MMP in rotenone-treated cells (FIG. 5B). Confocal microscopy identified depolarized mitochondria, which showed medium to low MitoTracker mitochondrial staining from normal mitochondria, showing that the mitochondrial membrane potential was not lost, thus showing stronger staining. Parkin and PINK1 levels were significantly increased in depolarized mitochondria (FIG. 5C and FIG. 5D). Overexpression of both Parkin and PINK1 increased the accumulation of LC3-II (FIG. 5E). These findings indicate that Parkin-mediated mitophagy occurs in RPE-MC cells.

2.6. 마이토파지는 RPE-MC 세포들의 세포 보호에 기여한다. 2.6. Mitophagy contributes to the cellular protection of RPE-MC cells.

Because autodigestion inhibitors reduce the viability of RPE-MC cells, mitophagy appears to be the basis for autodigestion-related protection of rotenone-treated RPE cells. To demonstrate that mitophagy plays a cytoprotective role, we tested the effect of Parkin depletion on cell death. Parkin knockdown partially prevented rotenone-induced increases at LC3-II levels (FIG. 6A). Notably, Parkin knockdown significantly increased RPE cell death induced by rotenone treatment alone (FIG. 6B). These findings indicate that mitophagy plays a role in the survival of RPE-MC cells. Transmission electron microscopy revealed the presence of degraded mitochondria and autophagosomes (autophagosomes) in rotenone-treated cells. Mitochondria swallowed with lysosomes were also observed in RPE-MC cells (Fig. 6Ca-Cd), indicating active mitophagy in RPE-MC cells. There were no autophagosomes or lysosomes observed in RPE-MC cells treated with rotenone and 3MA, in which the degraded mitochondria remained undigested (FIG. 6Cd). In RPE-MC cells treated with rotenone and Baf-A1 or chloroquine, various autophagosomes were present, and various degraded mitochondria were isolated from autophagosomes (Figure 6 Ce and Cf). These findings indicate that autodigestion inhibitors blocked the removal of degraded mitochondria in RPE-MC cells. These findings demonstrate that Parkin-mediated mitophagy is the basis for autodigestion-related cytoprotection of RPE-MC cells.

2.7. 알파 B 크리스탈린(crystallin)은 RPE-MC 세포를 세포자살로부터 보호한다. 2.7. Alpha B crystallin protects RPE-MC cells from apoptosis.

Since alpha B crystallin (αB-crystallin) is a major anti-apoptotic factor in RPE cells, we examined whether αB-crystallin has an anti-apoptotic effect in RPE-MC cells. αB-crystallin knockdown significantly increased the decrease in RPE-MC cell viability compared to cells treated with rotenone alone (FIG. 7A). These data show that αB-crystallin prevents RPE-MC cells from falling into cell death. In particular, TUNEL analysis showed that αB-crystallin knockdown induced apoptosis in RPE cells treated with autophagy inhibitors (FIG. 7B), indicating that RPE is susceptible to cell death due to inhibition of autodigestion. Cells show anti-apoptotic activity. Flow cytometry showed that αB-crystallin knockdown results in significantly increased accumulation of Annexin V-positive RPE-MC cells, indicating that αB-crystallin knockdown results in inhibition of autophagy of RPE-MC cells. It is shown that killing causes a change from non-apoptotic cell death to apoptotic cell death (FIG. 7C). These findings suggest that αB-crystallin exerts anti-apoptotic activity in RPE-MC cells, preventing them from experiencing apoptosis.

Claims

A pharmaceutical composition for preventing or treating proliferative vitreoretinopathy (PVR) containing a mitochondrial complex I inhibitor as an active ingredient.
The pharmaceutical composition of claim 1, wherein the mitochondrial complex I inhibitor is Rotenone.
The pharmaceutical composition of claim 1, wherein the active ingredient further comprises an autophagy inhibitor.
The pharmaceutical composition of claim 3, wherein the self-extinguishing inhibitor is selected from the group consisting of 3-methyladenine, bafilomycin A1, and chloroquine. Composition.
The pharmaceutical composition according to any one of claims 1 to 4, wherein the active ingredient modulates cell death of Retinal pigment epithelial (RPE) cells.
6. The pharmaceutical composition of claim 5, wherein the regulation of cell death induces apoptosis.