Use of a dusp6 inhibitor in manufacturing a medicament for rescuing retinal degeneration

Abstract

The present invention provides a method for rescuing retinal disease, comprising administering a pharmaceutical composition to an individual in need, the pharmaceutical composition comprising a DUSP6 inhibitor via the ERK1 / 2 autophagy pathway.

Description

Methods for Rescuing Retinopathy and Pharmaceutical Compositions The present invention relates to a method for rescuing retinopathy and a pharmaceutical composition. Retinopathy is the main cause of central vision loss in the elderly population [1, 2]. A systematic literature review further demonstrates the importance of this condition from a global perspective and predicts that its prevalence will gradually increase due to the exponential growth of the aging population [3]. Although aging is the main risk factor in the retinopathy process, it is also affected by other factors, including genetic susceptibility and oxidative stress. The retina has become a target for oxidative stress because it is constantly exposed to the light stimuli required for vision [4]. Oxidative stress occurs when the ROS level exceeds a certain threshold, which leads to the activation of protective mechanisms in cells, such as autophagy [5]. In addition, excessive ROS can disrupt autophagic homeostasis and damage the retina at the molecular level, thereby weakening its structural and functional integrity. Under normal circumstances, retinal pigment epithelial cells (RPE) play a key role in neutralizing harmful free radicals to maintain the redox homeostasis in the retina [4]. In addition, RPE phagocytoses toxic lipid-protein aggregates derived from the outer segment of photoreceptors via autophagy [6]. However, this continuous exposure to oxidative stress will ultimately cause these RPE to shift to the exhaustion stage, hindering their proper and normal functions [7]. Due to the harmful effects of oxidative stress on retinal structure damage, many researchers have used oxidative compounds that preferentially damage RPE, such as sodium iodate (NaIO 3 ), to generate in vitro and in vivo retinopathy disease models [8]. Autophagy is a catabolic process that removes unwanted or damaged cell components through lysosomal digestion. Interestingly, several evidences have shown that autophagy is closely associated with oxidative stress [9, 10]. Autophagy regulates cell production by recycling metabolic precursors and reduces oxidative stress by clearing intracellular toxic waste

[11] . As described, autophagy is crucial for cell homeostasis and the metabolism of toxic lipid-protein aggregates in retinal physiology. However, autophagy dysregulation is associated with an increased susceptibility to oxidative stress, which may ultimately lead to the progression of RPE lesions in AMD [12, 13]. Studies have shown that sodium iodate can induce autophagy in RPE cells, resulting in a decrease in mitochondrial activity through mitophagy, thereby affecting cell viability

[14] . Although autophagy has occurred, in NaIO 3Partial blockade of the autophagic flux was also found in the treated cells

[15] . Chan observed that inhibiting autophagy in RPE cells enhanced the susceptibility of the cells to NaIO 3 , while activation of autophagy could counteract the cell death mechanism induced by NaIO 3

[16] . The extracellular signal-related kinases (ERK1 / 2) are some of the main signaling pathways that induce and maintain autophagy

[17] and regulate the maturation of autophagic vacuoles

[18] . Specifically, there are three subfamilies in the ERK1 / 2 family: serine / threonine phosphatases (PP2A, PP2C), tyrosine phosphatases (STEP, HePTP, PTP-SL), and dual-specificity protein phosphatases (DUSP)

[19] . Among the DUSP family, DUSP6 specifically interacts with ERK1 / 2 and plays a balancing role in the downstream regulation of ERK1 / 2

[20] . Inhibition of DUSP6 has been shown to play an inhibitory role in some diseases such as gastric cancer or ovarian cancer

[21] . Previous studies have also reported the upregulation of DUSP6 in response to external factors including oxidative stress, and the involvement of autophagy proteins (ATG) in regulation through the ERK1 / 2 pathway in many cell types

[22] . Although the role of DUSP6 protein in ERK-mediated cell functions has been demonstrated in many cells, the relationship between DUSP6 and autophagy regulation in RPE cells in the case of oxidative stress-induced cell death remains unclear. Accordingly, there is a need to develop a method to ensure the rescue of retinal pigment epithelium. The present invention unexpectedly discovers that in in vivo and in vitro models of retinopathy, regulating dual-specificity protein phosphatase 6 (DUSP6) can rescue retinopathy via the ERK1 / 2 autophagy pathway. In one aspect, the present invention provides a method for rescuing retinopathy, which comprises administering to an individual in need a pharmaceutical composition comprising a DUSP6 inhibitor via the ERK1 / 2 autophagy pathway. In a specific embodiment of the present invention, the DUSP6 inhibitor is sodium iodate (NaIO 3 ) or (E / Z)-BCI hydrochloride (BCI). In an example, the present invention provides a method for treating retinopathy diseases, which comprises administering to an individual in need a pharmaceutical composition comprising a therapeutically effective amount of a DUSP6 inhibitor and a pharmaceutically acceptable carrier, wherein the DUSP6 inhibitor is sodium iodate or BCI. In one example of the present invention, the inhibitor is sodium iodate, which provides the effect of significantly increasing oxidative stress and autophagy. In another example of the present invention, the DUSP6 inhibitor is (E / Z)-BCI hydrochloride (BCI), which provides a significant increase in structural repair in the retinal layer of the individual. The present invention discovers that inhibiting DUSP6 activity can promote autophagy flux through the ERK pathway, which is via upregulating the immunoblot expression of sodium iodate (NaIO 3 ), as an oxidative stress inducer, in the ARPE-19 cell line and C57BL / 6N mice, showing a significant increase in autophagy flux activity. The present invention also discovers that BCI provides an effect on the regulation of the ERK1 / 2 pathway in a retinopathy model, especially significantly increasing autophagy flux in vitro and structural repair of the retinal layer in vivo. It can be concluded that DUSP6 inhibition promotes autophagy flux activity by upregulating the ERK cascade. The present invention demonstrates that the retinal layer is restored after treatment with the DUSP6 inhibitor NaIO 3 or BCI; it shows that the DUSP6 inhibitor can rescue retinal damage by restoring autophagy flux in the mouse retina. It shows that upregulating DUSP6 can lead to defective autophagy flux function in the RPE, and the DUSP6 inhibitor can restore autophagy induction. Accordingly, the DUSP6 inhibitor can be used as a potential treatment method for retinopathy diseases. In yet another aspect, the present invention also provides a pharmaceutical composition for treating retinopathy diseases, which comprises a therapeutically effective amount of a DUSP6 inhibitor and a pharmaceutically acceptable carrier. In yet another aspect, the present invention also provides the use of a DUSP6 inhibitor in the manufacture of a medicament for treating retinopathy diseases, or the use of a DUSP6 inhibitor in treating retinopathy diseases. In a specific embodiment of the present invention, the DUSP6 inhibitor is sodium iodate. In another specific embodiment of the present invention, the DUSP6 inhibitor is BCI. Those skilled in the art will understand the features and advantages of the present invention. Although those skilled in the art can make many changes, such changes fall within the scope of the present invention. When read in conjunction with the accompanying drawings, the foregoing invention content and the following embodiments of the present invention will be better understood. For the purpose of illustrating the present invention, the currently preferred specific embodiments are shown in the drawings. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a sample" will include a plurality of such samples and equivalents thereof known to those skilled in the art. As used herein, the term "individual" means a human or a mammal, such as a patient, a companion animal (e.g., a dog, a cat, etc.), a farm animal (e.g., a cow, a sheep, a pig, a horse, etc.) or a laboratory animal (e.g., a rat, a mouse, a guinea pig, etc.). As used herein, the term "carrier" means a material commonly used in the formulation of pharmaceutical compositions, which is used to enhance stability, sterility and delivery ability. When the peptide delivery system is formulated into a solution or suspension, the delivery system is in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as water, buffered aqueous solutions, 0.8% saline, 0.3% glycine, hyaluronic acid, etc. The composition may contain physiologically acceptable adjuvants as needed to approximate physiological conditions, such as pH regulators and buffers, tonicity regulators, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. As used herein, the term "pharmaceutically acceptable" means a substance that is free of excessive toxicity, irritation, allergic reaction or other problems or complications and is suitable for use in humans and / or animals. As used herein, the term "therapeutically effective amount" means an amount that treats an individual with a reasonable benefit-risk ratio applicable to any medical treatment, which means an amount sufficient to confer a therapeutic effect. In particular, a "therapeutically effective amount" means an amount used to improve symptoms associated with a disease, prevent or delay the onset of a disease and / or reduce the severity of the symptoms of a disease. A therapeutically effective amount is usually administered in a dosage regimen that may include multiple or unit doses. The therapeutically effective amount (and / or the appropriate unit dose in the effective dosage regimen) will depend on, for example, the route of administration, the combination with other agents. The therapeutically effective amount will vary according to the age, weight, general health, gender and diet of the individual to be treated, the time of administration, the route of administration, the duration of treatment and various factors, including similar factors well known in the art. As used herein, the term "treatment" (also referred to as "treatment" or "disposal") means a specific disease or disorder, including partially or completely alleviating, improving, relieving, suppressing, delaying the onset of one or more symptoms or diseases, and reducing the severity of one or more symptoms or diseases. According to the present invention, a method for rescuing or treating retinopathy diseases comprises administering to an individual in need thereof a pharmaceutical composition comprising a DUSP6 inhibitor via the ERK1 / 2 autophagy pathway. In some examples of the present invention, the DUSP6 inhibitor is sodium iodate (NaIO 3 ) or (E / Z)-BCI hydrochloride (BCI). As used herein, terms such as "retinopathy disease", "retinopathy", or "pathological retinopathy disease" refer to progressive neurological diseases caused by gene mutations and / or environmental or pathological damage to the retina, which are incurable so far. In one example of the present invention, the DUSP6 inhibitor is sodium iodate, which has been confirmed to show a significant increase in oxidative stress and autophagy. In another example of the present invention, the DUSP6 inhibitor is (E / Z)-BCI hydrochloride (BCI), which has been confirmed to provide a significant promotion of structural repair in the retinal layers of the individual. According to the present invention, DUSP6 inhibitors (such as sodium iodate or BCI) can be used as potential treatment methods for retinopathy diseases, which, by upregulating DUSP6, can lead to dysfunction of the autophagic flux of retinal pigment epithelium (RPE) and restore autophagy induction. The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration and not limitation. Examples Materials and Methods 1.1 Cell Lines and Cell Culture The ARPE-19 cell line was purchased from ATCC (ATCC® CRL-2302™). The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (50 U / mL). The cells were subcultured at 37°C and 5% CO 2 when they reached 90% confluence, with a relative humidity of 95%. 1.2. Sodium Iodate and Protein Regulators The treated ARPE-19 cells were seeded in 6 cm culture dishes at a density of 2×10 6 cells for 24 hours and treated with sodium iodate (NaIO) at concentrations of 0, 2, 4, and 8 mM at 37°C 3) (Sigma, Saint Louis, MO, USA) for 6 hours. Cells were treated with 75 nM of the autophagy inhibitor bafilomycin A1 (Sigma, Saint Louis, MO, USA) or with 1.25 µM or 2.5 µM of the DUSP6 inhibitor BCI (Sigma, Saint Louis, MO, USA) at 37 °C for 6 hours. Proteins were collected after treatment for further study. 1.3 Immunoblot analysis C57BL / 6N mice were sacrificed and their eyeballs were removed. Excess muscle tissue outside the sclera was removed, and a hole was punched in the cornea with a needle, and then the entire cornea was removed. Next, the inventor removed the lens and retina, and finally lysed the remaining tissue in RIPA buffer (10× Merck Millipore, Temecula, CA, USA) containing 1% protease inhibitor. At the same time, ARPE-19 cells were collected in vitro and lysed in RIPA buffer containing 1% protease inhibitor. Subsequently, the cell lysates were centrifuged at 4°C for 10 minutes and the supernatants were collected. Then, the protein concentration was measured via a BCA protein quantification kit. Equal weights of total protein were electrophoretically separated on SDS / PAGE. After transferring the protein to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA), the membrane was incubated with blocking buffer (1× TBST and 5% non-fat milk) for 1 hour at room temperature. After the blocking step, the membrane was incubated overnight at 4°C with the following primary antibodies: DUSP6 (Abcam, Cambridge, UK), Phospho-ERK1 / 2 (Cell Signaling, Danvers, MA, USA), ERK1 / 2 (Cell Signaling, Danvers, MA, USA), LC3B (Novus, Centennial, CO, USA), SQSTM1 / p62 (Abcam, Cambridge, UK), LAMP2 (Abcam, Cambridge, UK), Beclin-1 (Cell Signaling, Danvers, MA, USA), and β-actin (Sigma, Saint Louis, MO, USA). Next, the membrane was washed 3 times with TBST and incubated with HRP-conjugated secondary antibodies (including goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) and goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA)) for 1 hour at room temperature. The antigen-antibody complexes were detected via an enhanced chemiluminescence (ECL) substrate kit (Bioman, Millipore, Bedford, MA, USA). The bands were analyzed and quantified by ImageJ (NIH, Bethesda, MD, USA) analysis software. 1.4. Autophagy flux assay Cell autophagy flux was measured via an autophagy detection kit (Abcam, Cambridge, UK). First, the cells were pretreated with an autophagy inducer and chloroquine for 16 hours as a positive control. After NaIO 3After treatment, the cells were washed twice with 1× assay buffer and 100 µL of Microscopy Dual Detection Reagent, which consists of a green detection reagent and a nuclear staining buffer, was added. The samples were protected from light and incubated at 37°C for 30 minutes. Subsequently, the cells were carefully rinsed with 100 µL of 1× assay buffer and incubated with 4% formaldehyde for 20 minutes. After washing three times with 1× assay buffer, the stained cells were analyzed by wide-field fluorescence microscopy. 1.5. Quantitative real-time polymerase chain reaction (qRT-PCR) The cDNA was diluted to 200 ng / µL with sterile water and mixed with SYBR PCR Master Mix and primers. The amplification reaction was carried out in a thermal cycler according to the manufacturer's instructions. The mRNA abundance was quantified using the threshold cycle method. The mRNA expression of GAPDH was used as a normalized internal control. 1.6. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay The MTT assay kit (Sigma, Saint Louis, MO, USA) was used for cell viability testing. ARPE-19 cells were seeded at 5×10 4 cells per well in a 96-well plate for 24 hours and treated with different concentrations of NaIO 3 (0, 2, 4, 8, 16, 32 mM) for 6 hours. After treatment, the cells were washed with PBS and the inventors added 10 µL of MTT working solution (5 mg / mL, dissolved in phosphate buffer solution, Sigma, Saint Louis, MO, USA) and incubated in a CO 2 incubator at 37°C for 4 hours. After incubation, 50 µL of DMSO (solubilizer) was added to each well and then mixed with a micropipette. The presence of live cells was observable as a purple color due to the formation of formazan crystals. Finally, the intensity was measured by reading the OD540 on a microplate spectrophotometer (Tecan Austria GmbH 5082, Grobdig, Austria). 1.7. ROS detection assay (DHR-123 assay) Intracellular ROS was measured by the fluorescent probe dihydro-rhodamine 123 (DHR-123) (Invitrogen, Carlsbad, CA, USA) to examine that DHR-123 turns into rhodamine 123 (Rh-123) when oxidized by ROS. First, ARPE-19 cells were seeded at 5×10 5 cells per well in a 24-well plate for 24 hours. Sodium iodate (NaIO 3 ) was dissolved in dimethyl sulfoxide (DMSO). After treatment with different concentrations (0, 2, 4, 8 mM) of NaIO 3 for 6 hours, the cells were cultured with 2 μM DHR-123 dye in the dark at 37°C in 5% CO 2 with 95% relative humidity for 30 minutes. Subsequently, the fluorescence intensity of Rh-123 was measured by a fluorescence microscope (Olympus America, Melville, NY, USA) at an excitation wavelength of 488 nm. 1.8. Animals Healthy male C57BL / 6N mice (8 weeks old, approximately 20 g) were purchased from LASCO. The mice were housed under standard conditions of a 12:12 hour dark-light cycle and had free access to standard rodent food and water. For each experimental group, experiments were performed on 3 mice. All mice were treated in accordance with the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) statement on the use of animals in ophthalmic and vision research, and the animal study was approved by the Institutional Animal Care and Use Committee of Taipei Veteran General Hospital (IACUC), approval number: 2020-038 (January 1, 2020). 1.9. Establishment of a retinopathy model by treating sodium iodate via intraperitoneal injection The sodium iodate solution (Sigma, Saint Louis, MO, USA) was diluted with PBS for in vivo AMD simulation model. The final concentration for mouse treatment was 30 mg / kg, and they were administered via intraperitoneal injection. Subsequently, the mice were sacrificed, and the eyeballs of mice at different stages were collected for further histological analysis. According to the established model, the protective effect of the DUSP inhibitor will be confirmed by intravitreal injection and real-time imaging observation. 1.10. Histological evaluation of the retina Retinal samples were fixed overnight with 4% paraformaldehyde (Sigma, Saint Louis, MO, USA), followed by 1× PBS. Fixation, dehydration, washing, infiltration, and embedding were performed according to the method of Bio-Check Laboratories. Briefly, each retinal sample was cut into 3-μm sections using a vibratome (Leica, Buffalo Grove, IL, USA), and then hematoxylin and eosin (H&E) staining was performed. Retinal morphology was observed using an optical microscope (Olympus America, Melville, NY, USA). Subsequently, retinal thickness was measured by Image J (NIH, Bethesda, Maryland, USA), which was defined as the distance between the inner limiting membrane, Bruch’s membrane, and the outer nuclear layer (ONL). 1.11. Fundoscopy and Optical Coherence Tomography According to previous reports, after anesthesia with a mixed solution of Zoletil (50 mg / kg) and Ropum (10 mg / kg), images of the mouse retinal structure were obtained by multi-contrast optical coherence tomography (OCT)

[23] . Fundus photographs were acquired using a retinal imaging camera (Nikon, Shinagawa, Tokyo, Japan). Fundoscopy and OCT images were captured at the exact retinal space around the optic nerve. 1.12. Immunofluorescence staining In vitro, cells were seeded in 24-well plates at a density of 2×10 5 cells per well and cultured at 37°C with 5% CO 2 for 24 hours. After treatment with 4 mM NaIO 3After treatment for 6 hours, the cells were washed twice with PBS and then fixed with 4% paraformaldehyde (PFA) (Sigma, Saint Louis, MO, USA) for 15 minutes. After washing three times with PBS, the cells were permeabilized and blocked with a blocking solution (0.3% BSA and 0.1% Triton X-100 in PBS) for 1 hour at room temperature. After blocking, the cells were incubated with primary antibodies overnight at 4°C. The primary antibodies included DUSP6 (Abcam, Cambridge, UK), Phospho-ERK1 / 2 (Cell Signaling, Danvers, MA, USA), LC3B (Novus, Centennial, CO, USA), and SQSTM1 / p62 (Abcam, Cambridge, UK). After incubation, the cells were washed three times with PBST (0.1% Triton X-100 in PBS). Secondary antibodies (1:700) were applied for 3 hours at room temperature. The secondary antibodies included IgG Alexa 488 goat anti-rabbit IgG and Alexa 594 goat anti-mouse IgG. The cell nuclei were stained with DAPI (1:1000) for 10 minutes at 4°C, and the slides were observed with an optical microscope (Olympus America, Melville, NY, USA). In vivo, the sample sections were pretreated overnight at room temperature with a bleaching solution (1% NaH 2 PO 4 and 2.5% H 2 O 2 ) and then incubated with primary antibodies (including DUSP6, Phospho-ERK1 / 2, LC3B, SQSTM1 / p62, and RPE65) overnight at 4°C. After incubation, the samples were washed three times with PBST. The secondary antibodies (1:700) were applied for 1 hour at room temperature. The secondary antibodies included IgG Alexa 488 goat anti-rabbit IgG and Alexa 594 goat anti-mouse IgG. After treatment with a mounting medium, the slides were observed with a fluorescence microscope (Olympus America, Melville, NY, USA). 1.13. DUSP6 Inhibitor Treatment in Mice The mice were divided into three groups: a control group, a NaIO 3 group, and a BCI group. The BCI group of mice was given a dose of 30 mg / kg of NaIO 3, followed by intravitreal injection of BCI (0.5 mg / kg) for 3 days. All healthy control groups received intraperitoneal placebo injection, and the NaIO 3 group received intravitreal placebo injection. Retinal structure was analyzed by real-time imaging and H&E staining. Proteins were collected from the retina for further research. 1.14. Statistical analysis Experimental data are expressed as mean ± SEM. The standard distribution of the data was determined by the Kolmogorov–Smirnov test, and non-parametric values were analyzed using the Mann–Whitney test. For statistical analysis, one-way analysis of variance (ANOVA) and Student's t-test were performed; p < 0.05 was considered significant, and if p < 0.01, highly significant differences in the statistical data were accepted. 2. Results 2.1. Sodium iodate disrupts autophagic flux in ARPE-19 cells Dysfunction of the RPE is one of the main pathological features of retinopathy, and autophagy is an important lysosomal degradation process that can remove damaged organelles and misfolded proteins in the retina. However, the mechanism of retinopathy remains unclear, and little is known about whether autophagy plays a role in it. To investigate the pathological mechanism of retinopathy, ARPE-19 cells widely used in retinal pathology research were exposed to the oxidative inducer sodium iodate (NaIO 3 ) to establish a retinopathy model. To evaluate the expression of autophagy markers in RPE cells under the stress shock induced by NaIO 3 , ARPE-19 cells were treated with different concentrations of NaIO 3 (0, 2, 4, 8 mM) for 6 hours (Figure 1(a)). The inventors observed that NaIO 3 induced the generation of autophagosomes in a dose-dependent manner (Figure 1(b)). To further examine the expression levels of autophagy markers under NaIO 3 treatment, total proteins were collected and immunoblot analysis was performed. As shown in Figure 1c, autophagy markers (including LC3B-II, Beclin-1, and LAMP2) were gradually upregulated in cells treated with NaIO 3 . In addition, in NaIO 3An increase in LC3B-positive puncta was also observed in the treatment group, indicating autophagosome generation (Figure 1(d)). Their data indicated that NaIO 3 Effect on inducing autophagy in RPE cells. Based on immunoblot and immunofluorescence staining data, the inventors found that, in NaIO 3 treatment, the expression of the autophagy marker p62 (p62 / SQSTM1) increased. The p62 protein is a well-known substrate of selective autophagy, which interacts with LC3B, leading to the degradation of ubiquitinated substrates, followed by degradation in autophagosomes. However, it has been previously shown that the accumulation of p62 is associated with a lack of autophagic flux under oxidative stress

[24] . To further examine the autophagic flux in RPE cells, the autophagy inhibitor bafilomycin A1 (Baf-A1) was used to evaluate the cellular response in ARPE-19 cells treated with NaIO 3 treatment. Treatment with Baf-A1 led to a dose-dependent increase in the expression of LC3B-II, indicating that autophagy was in an early stage (Figure 1(e)). At the same time, the p62 expression level remained unchanged despite treatment with Baf-A1. In addition, compared with control ARPE-19 cells, any given NaIO 3 treatment dose did not affect the p62 mRNA level (Figure 1(f)). Their results indicated that the elevated p62 expression induced by NaIO 3 was not due to proteasome activity disruption or p62 gene upregulation. Therefore, NaIO 3 might have the effect of blocking the late autophagic flux in ARPE-19 cells. 2.2. Treatment with NaIO 3 induced DUSP6 and MAPK upregulation. In a retinopathy model induced by exposing ARPE-19 cells to oxidized low-density lipoprotein (oxLDL), it was found that DUSP6 was elevated in response to oxLDL treatment

[25] . However, the treatment effect of NaIO 3 on DUSP6 expression and the role of DUSP6 in the autophagic flux of retinopathy remained unclear. Therefore, the inventors attempted to study the expression of DUSP6 and its role in NaIO 3Role in the autophagy flux of RPE cells under treatment. Immunoblot assays were performed to examine the expression of DUSP6 and the regulated kinase (p-ERK). The results showed that the expression of DUSP6 was upregulated in a dose-dependent manner by treatment with NaIO 3 , and the p-ERK / ERK ratio representing ERK activity also increased (Figure 2(a)). Their data indicated that the DUSP6 and ERK pathways were stimulated by NaIO 3 treatment in ARPE-19 cells. Collectively, their data suggested that DUSP6 expression was induced by increased ERK activity in ARPE-19 cells under NaIO 3 treatment. 2.3. BCI promotes autophagy flux via ERK pathway activity in ARPE-19 cells To investigate whether inhibiting DUSP6 activity could regulate autophagy flux and counteract oxidative stress, the inventors cultured ARPE-19 cells with NaIO 3 for 6 hours in the presence or absence of BCI (a DUSP6 inhibitor). Subsequently, the inventors performed autophagy flux analysis by immunoblot assay and immunocytochemical analysis. After BCI treatment, the p-ERK / ERK ratio representing ERK activity increased significantly in a dose-dependent manner in cells treated with NaIO 3 (Figure 3(a)). Consistently, immunofluorescence staining showed that the puncta of p-ERK increased after BCI treatment regardless of the presence or absence of NaIO 3 (Figure 3b). This result indicated that BCI stimulated p-ERK signaling under both normal and NaIO 3 treatment conditions. In addition, the later stages of autophagy flux were examined using immunoblot and immunocytochemical staining. Immunoblot showed that autophagy markers were upregulated in a dose-dependent manner after BCI treatment (Figure 3c). The LC3B-II / LC3B-I ratio is shown in Figure 3d, and the quantification of p62 is shown in Figure 3e. In addition, fluorescence images showed the aggregation of autophagosomes in the BCI treatment group. Moreover, punctate perinuclear p62 representing strong autophagic activity was significantly upregulated in cells co-treated with NaIO 3 and BCI (Figure 3(f)). In addition, to evaluate autophagic flux in ARPE-19 cells treated with BCI, the inventors introduced the GFP-LC3-RFP-LC3∆G gene into ARPE-19 cells and monitored real-time images taken after BCI treatment. The inventors observed that the number of GFP-positive puncta increased under oxidative stress induced by NaIO 3 which presented a low autophagic flux. Interestingly, treatment with BCI led to a change in the GFP-positive signal to an RFP-positive signal, which indicated the induction of late autophagy by BCI treatment (Figure 3g). Next, qPCR analysis showed that the mRNA level of p62 was upregulated in the BCI group, which indicated that BCI could promote autophagy gene expression in RPE cells under oxidative stress, showing that BCI upregulated the p62 mRNA level under oxidative stress. ARPE-19 cells were co-treated with BCI and NaIO 3 for 6 hours, and the mRNA level of p62 was detected by qPCR. The positive effect of BCI on autophagy proteins indicated that BCI effectively promoted autophagic flux by blocking DUSP6 activity to stimulate the p-ERK-mediated autophagy pathway. In summary, those results showed that pharmacological activation of p-ERK by BCI could accelerate autophagosome formation and degradation of oxidative waste in response to oxidative stress induced by NaIO 3 2.4. Upregulation of the DUSP6 / p-ERK axis in NaIO 3 -induced in vivo retinopathy NaIO 3 has been widely used to induce RPE lesions in vivo due to its targeting of oxidative damage to the RPE

[26] . However, the pathogenesis of RPE lesions in the oxidative model induced by NaIO 3 remains unclear. To investigate the relationship between autophagy regulation and oxidative stress in vivo, the inventors intravenously injected wild-type C57BL / 6 mice with NaIO 3 and then performed retinal analysis by ultra-high-resolution multi-contrast optical coherence tomography (OCT)

[23] imaging and histological sections of cadaver tissues for 1 week (Figure 4(a)). Abnormal deposition of migrating cells in the RPE layer (red arrow) was observed 4 days after injection, and they worsened within 7 days (Figure 4(a)). The retina became thinner, and the boundaries between the layers were unclear. In addition, the boundary between the outer nuclear layer (ONL) and the inner nuclear layer (INL) almost disappeared (Figure 4(b)). The NaIO was further evaluated by hematoxylin and eosin (H&E) staining of retinal tissues 3 Effect on retinal structure. In the control group of mice treated with saline, the retina showed well-organized and clear boundaries of retinal layers, including the ganglion cell layer (GCL), INL, and ONL. The retinal pigment epithelium (RPE) layer was smooth and had uniform pigmentation, indicating that the cells were in a healthy state (Figure 4(c)). One day after injection with NaIO 3 , the retinal layers still remained regular and showed a slightly thicker melanin pattern (Figure 4(c)). Notably, four days after NaIO 3 injection, the arrangement of the ONL was significantly disordered. In addition, the uneven pigment deposition in the RPE layer indicated the lesion of the RPE (Figure 4(c)). Seven days after injection, the inner segment / outer segment junction showed severe damage above the RPE layer. At the same time, due to severe melanin hyperplasia, the migration of RPE cells was clearly observed (Figure 4(c)). In addition, due to the severe lesions of photoreceptors and the RPE layer, the retina became significantly thinner (Figure 4(c)). In summary, this histopathological study confirmed that NaIO 3 injection disrupted the arrangement of retinal layers and induced severe pigment deposition, accompanied by the accumulation of migratory cells and cell debris. The tight junction marker ZO-1 was used to verify cell-cell junctions before and after NaIO 3 treatment. The inventors' results showed that compared with the control group, cell-cell junctions were damaged after treatment with NaIO 3 indicating that cell-cell junctions were damaged after treatment with NaIO 3 and co-treatment with BCI rescued cell-cell junctions between RPE cells. By performing histological examination of eyeball specimens, the inventors found that after treatment with NaIO 3 the RPE layer was swollen and pigment cells had migrated to the OS layer. Based on previous studies, the inventors believed that cell aggregation was caused by the proliferation of microglial cells, and the inventors' data showed Iba1 expression between the ONL layer and the RPE layer, indicating immunohistochemical examination of Iba1 expression in the retina of mice treated with NaIO 3 In 30 mg / kg NaIO 3Representative immunohistochemical staining of Iba-1 and DAPI 7 days after injection [27,28]. They found that a retinopathy disease model was successfully established. To determine the role of DUSP6 in response to oxidative stress in this retinopathy model, the inventors collected the eyes of NaIO 3 -treated mice at different time points and detected the expression of DUSP6 protein in the retina after NaIO 3 treatment. Immunoblotting showed that DUSP6 was upregulated at 1 day and 7 days after NaIO 3 treatment. The expression of phosphorylated ERK was also significantly upregulated, especially at 7 days after injection (Figure 4(d)). The quantitative relationships of the expressions of DUSP6, ERK, and phosphorylated ERK are shown in Figures 4(e) and 4(f). In short, the inventors confirmed that the DUSP6 / ERK axis was upregulated in the mouse retina under oxidative stress induced by NaIO 3 and that this axis may play a key role in the regulatory mechanism of retinopathy. 2.5. Increased expression of autophagy markers in the retinopathy model In addition, to verify the autophagic flux in the in vivo NaIO 3 -induced retinopathy model, immunoblotting and immunofluorescence staining were performed. Immunoblot analysis showed that the autophagy markers LC3B-II, p62, and Beclin-1 were all upregulated after the NaIO 3 treatment process (Figure 5(a)). Quantitative data showed that the expression of LC3B-II was significantly increased under the oxidative stress induced by NaIO 3 (Figures 5(b), 5(c)). This result indicates the accumulation of autophagosomes, which证实 the activation of autophagic flux. The upregulation of p62 expression further indicates the characteristics of autophagic flux (Figures 5(a), 5(c)). In addition, immunofluorescence staining showed that LC3B was increased in different retinal layers of NaIO 3 -treated mice, while the upregulation of p62 was mainly found in the photoreceptor and RPE layers (Figure 5(d)). Their results showed that NaIO 3 could effectively promote the autophagic flux in the retina (especially in RPE cells). 2.6. BCI activates autophagic flux by upregulating via the ERK pathway in vivo Based on the inventors' observations, using BCI to inhibit DUSP6 activity can activate autophagy flux via upregulation of in vitro 62 mRNA expression. To confirm the protein diversification and signal patterns in the mouse retina after BCI treatment under NaIO 3 induction, retinal sections were analyzed by immunofluorescence staining. The inventors found that phosphorylated ERK spots increased around the ONL layer and RPE layer under oxidative conditions and were significantly enhanced after BCI treatment (Figure 6(a)). This result indicates that BCI upregulates ERK activity in the mouse retina, especially in photoreceptor and RPE cells. In addition, excessive co-localization of LC3B and p62 was observed between the ONL layer and the RPE layer in the BCI treatment group, indicating higher autophagy activity in photoreceptor and RPE cells (Figure 6(b)). In short, their findings indicate that under oxidative stress, treating with BCI induces phosphorylated ERK activity and autophagy flux in the mouse retina, especially in photoreceptor and RPE cells. To further investigate the protective effect of treating BCI on NaIO 3 induced oxidative damage in vivo, the inventors performed histological examinations and showed that after melanin deposition, the ONL boundary was significantly disordered (Figure 6c, red arrow). Migratory cells were observed in the ONL of the NaIO 3 treatment group, indicating loss of cell-cell connections in the RPE under oxidative stress (Figure 6(c)). In the BCI group, the ONL disorder was alleviated by treating with BCI in the presence of NaIO 3 induced oxidative stress. Compared with the mouse retina treated only with NaIO 3 treatment, melanin deposition on the retina was also significantly reduced (Figure 6(c)). In addition, immunofluorescence staining showed that treating BCI restored the ONL thickness, which decreased due to thinning of the ONL after NaIO 3 treatment. Their results indicate the protective effect of BCI in retinal cells (Figure 6(d), 6(e)). In summary, the inventors' results show that under NaIO 3 induced oxidative stress, inhibiting DUSP6 prevents retinal lesions by promoting autophagy flux. 3. Discussion The irreversible progression of retinopathy involves congenital and acquired traits and ultimately leads to cell death in the RPE layer, which may result in blindness. Age-related macular degeneration (AMD) is the most common cause of retinopathy, characterized by RPE cell lesions (dry AMD) or choroidal neovascularization (wet AMD)

[29] . In addition, other factors, including oxidative stress, inflammation, and hypoxia, can also cause retinopathy

[13] . Exposure of RPE cells to oxidative stress leads to RPE cell death and encompasses the pathogenesis of AMD as a disease model. However, the underlying molecular mechanisms contributing to the progression of this disease, such as ROS-mediated signaling pathways, mitochondrial function, and autophagy, remain to be investigated. To answer this question, the inventors established a retinopathy model using NaIO 3 ; an oxidative inducer on the RPE cell line ARPE-19; and a mouse model C57BL / 6N. The mouse model received a single intraperitoneal injection of 30 mg / kg of NaIO 3 . Their results showed that treatment with NaIO 3 caused extensive damage to RPE cells and disrupted the retinal structure in mice, especially the RPE layer. Autophagy-related proteins play a key role in autophagy as they are the most tightly regulated components of this pathway. Beclin-1 regulates autophagy initiation and the lipidated form of microtubule-associated protein light chain 3-II (LC3-II)

[29] . The lipidated form of microtubule-associated protein light chain 3-II (LC3-II) has been shown to be a reliable marker of intact autophagosomes. In addition, LAMP2 plays a key role during lysosome formation. The inventors' immunoblot data confirmed that autophagy was enhanced at multiple levels. First, the increased protein expression of Beclin-1, LC3-II, and LAMP2 indicated an increase in early autophagic flux in RPE cells under oxidative stress. Second, the phosphorylation of ERK protein in RPE cells treated with NaIO 3 was higher than that in control RPE cells, indicating that the RPE underwent autophagic damage. Notably, in the present invention, it was confirmed that DUSP6 acts as a negative regulator of the ERK pathway by reducing NaIO 3Autophagy response in the treated RPE cells. A further novel finding of the present invention is that the inhibition of DUSP6 activity by BCI can promote autophagic flux and restore retinal structure. Inhibition of autophagic flux is typically associated with ubiquitin-positive aggregates or inclusion bodies and with large perinuclear aggregates

[31] . Impairment of the autophagy process has also been demonstrated in other neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease

[32] . The protein p62, also known as sequestosome 1 (SQSTM1), is a protein that is itself degraded by autophagy and can act as a link between ubiquitinated proteins and the autophagy machinery, thereby promoting their degradation in lysosomes. p62 accumulates when autophagy is inhibited and the level of p62 decreases when autophagy is induced, indicating that p62 can be used as an autophagic flux reporter. The planned comparison shows that the accumulation of p62 in the retinas of mice treated with BCI is significantly reduced, indicating the restoration of autophagic flux in RPE cells. The mitogen-activated protein kinase (MAPK) pathway converts signals from mitogens into signals that regulate transcription and affects cell proliferation, differentiation, and apoptosis by activating a protein kinase cascade

[33] . Extracellular signal-regulated protein kinase (ERK1 / 2) is one of the members of the MAPK family and plays a key role in transmitting extracellular signals to the nucleus and regulating cell proliferation, cell differentiation, and the cell cycle

[34] . Studies have shown that ERK1 / 2 is closely related to the regulation of the visual cycle and plays a key role in the survival of RPE and photoreceptor cells

[35] . It has been shown that the MAPK / ERK signaling pathway may be involved in the reduction of autophagic activity of RPE cells on nitrite-modified extracellular matrix (ECM). In the present invention, NaIO 3 The regulation of induced autophagy was found to occur in an ERK1 / 2-dependent manner. The appearance of this result is consistent with previous literature, and the inventors found that autophagy is involved in the upregulation of the stress proteins LC3B and p62 and the activation of the ERK pathway induced by NaIO 3 Their data show that the ERK1 / 2 pathway activates autophagic flux, which contributes to a possible survival mechanism under oxidative stress conditions. The present invention found that NaIO 3The induced ERK / p-ERK pathway can upregulate the expression level of DUSP6 in RPE cells, and inhibiting DUSP6 can rescue impaired autophagy. Their findings indicate the regulatory mechanism of the DUSP6 / ERK axis in retinal cells. To further confirm the role of DUSP6 in ERK-mediated autophagy in retinal cells, BCI was used to inhibit DUSP6 activity and promote autophagic flux. Interestingly, it was found that the p-ERK / ERK ratio increased significantly after BCI treatment, along with the upregulation of autophagy markers in a dose-dependent manner. In addition, treating with BCI led to an increase in the amount of punctate p62 around the nucleus in RPE cells under oxidative stress. Furthermore, compared with cells treated only with NaIO 3 treatment, the p62 mRNA level was upregulated after BCI treatment. According to their findings, other autophagy markers, including LC3B, Beclin-1, and Lamp2, were also upregulated in NaIO 3 -treated cells due to BCI treatment. By real-time imaging of RPE cells treated with NaIO 3 in a retinal lesion model, their data were further confirmed. The data confirmed that LC3B-II increased significantly after BCI treatment, indicating the inhibition of autophagic flux by DUSP6 under oxidative stress conditions. Their results suggest that BCI may counteract the harmful effects of oxidative stress in RPE cells through ERK1 / 2-mediated upregulation of autophagy, and provide clues for the development of new mediating strategies during the disease state by restoring autophagic flux and preventing oxidative stress-induced RPE cell dysfunction abnormalities. 5. Conclusion. In summary, the results of this study confirmed that NaIO 3It is an oxidant that can activate the ERK1 / 2 pathway and regulate DUSP6, resulting in the suspension of autophagy flux in RPE cells. Treating with the DUSP6 inhibitor BCI can regulate autophagy activity and provide a novel therapeutic agent that helps restore autophagy flux and prevent oxidative stress-induced retinopathy (Figure 7). References 1. 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[CrossRef]. None The foregoing invention content and the following embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, the currently preferred specific embodiments are shown in the drawings. The drawings include: Figure 1 shows that sodium iodate disrupts autophagic flux in ARPE-19 cells. (a) Schematic diagram of the NaIO 3 treatment protocol. (b) Detection of autophagosomes in ARPE-19 cells treated with NaIO 3 using an autophagy detection kit. Scale bar = 100 µm. (c) Representative dot blot analysis of autophagy-related protein expression after NaIO 3 treatment. Quantitative analysis of immunodots. (d) Immunocytochemical staining of LC3B and p62 in ARPE-19 cells after NaIO 3 treatment. The cell nuclei were stained with DAPI. Scale bar = 50 µm. (e) Representative dot blot analysis of autophagy flux-related protein expression under Baf-A1 treatment. (f) Real-time quantitative PCR showing the mRNA level of p62; n = 3. Representative data from three independent experiments are shown. * p < 0.05 and ** p < 0.01. Figure 2 shows the upregulation of DUSP6 and MAPK after NaIO 3 treatment. (a) Representative dots of DUSP6 / p-ERK axis expression and quantitative analysis of DUSP6 expression level in ARPE-19 cells after NaIO 3 treatment. (b) Quantitative analysis of p-ERK level. Representative data from three independent experiments are shown. * p < 0.05 and ** p < 0.01. Figure 3 shows that BCI promotes autophagic flux via the ERK pathway in ARPE-19 cells. (a) Representative immunoblot analysis of p-ERK / ERK axis expression in ARPE-19 cells treated with 1.25 and 2.5 μM BCI under oxidative conditions. (b) Immunocytochemical staining shows p-ERK signals in ARPE-19 cells with or without BCI treatment under oxidative conditions. Scale bar = 50 μm. (c) Representative immunoblot analysis shows the expression of autophagy-related proteins in AR-PE-19 cells treated with 1.25 and 2.5 μM BCI under oxidative conditions. (d, e) Quantitative analysis of immunoblots. Representative data from three independent experiments are shown. * p < 0.05. (f) Immunocytochemical staining shows the expression levels of LC3B and p62 in ARPE-19 cells treated with BCI (1.25 μM) under oxidative conditions. Scale bar = 50 μm. (g) Real-time images of the expression levels of full-length LC3B (GFP) and cleaved LC3B (RFP). The right subgraph represents the image of the white boxed area in the left subgraph with high magnification. Scale bar = 20 μm. Figure 4 shows that the DUSP6 / p-ERK axis is upregulated in NaIO 3 -induced in vivo retinopathy. (a) Schematic diagram depicting drug treatment and tissue preparation. (b) Immediate fundus and retinal structure at different times after NaIO 3 injection were observed by fundus microscopy and OCT system. Red arrows indicate irregular structures on the RPE layer. Representative data from three independent experiments are shown. (c) H&E staining shows the retinal structure of mice treated with NaIO 3 . Abnormal deposits on the RPE layer are indicated by red arrows. Scale bar = 50 μm. (d) Representative blots show the expression levels of the DUSP6 / p-ERK axis in C57BL / 6 mice after NaIO 3 injection. (e, f) Quantitative analysis of (d). * p < 0.05. Figure 5 shows that autophagy-related genes are upregulated in in vivo NaIO 3 -induced retinopathy. (a) Representative blots show autophagy-related markers in C57BL / 6 mice after NaIO 3 injection. (b, c) Quantitative analysis by Western blotting. * p value < 0.05 compared to the control group. (d) Immunofluorescence staining was used to observe the administration of NaIO 3 LC3B and p62 expression levels in the injected mouse retina. Scale bar = 50 µm. Figure 6 shows that BCI activates autophagic flux by upregulating the ERK pathway in vivo. (a) Immunofluorescent staining of p-ERK in the mouse retina treated with BCL. 50 µm. (b) Immunofluorescent staining of LC3B and p62 in the mouse retina treated with BCL. 50 µm. (c) The morphology of the mouse retina after treatment with BCL was confirmed by H&E staining and (d) immunofluorescent staining. The red bar shows the thickness of the ONL layer. Scale bar = 50 µm. (e) The retinal thickness was calculated using Image J. *Compared with the NaIO 3 group, p < 0.05. Figure 7 illustrates the conclusion, which shows that inhibiting DUSP6 protects RPE and retina from NaIO 3 induced oxidative stress-mediated autophagy dysfunction involving the ERK signaling pathway. None

Claims

1. The use of a DUSP6 inhibitor in the manufacture of an agent for rescuing retinopathy, wherein the retinopathy is rescued via the ERK1 / 2 autophagy pathway, and the DUSP6 inhibitor is (E / Z)-BCI hydrochloride (BCI).

2. As claimed in claim 1, wherein the DUSP6 inhibitor provides a significant increase in oxidative stress and autophagy.

3. As claimed in claim 1, wherein the DUSP6 inhibitor provides a significant increase in structural repair in the retinal layer.

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