TWI775101B - Use of emodin in manufacture of medicament for treating retinal ischemia or a disease, condition, or disorder associated with retinal ischemia - Google Patents

Abstract

The present invention relates to a use of emodin in manufacture of medicament for treating retinal ischemia, or a disease, condition, or disorder associated with retinal ischemia in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of emodin or a composition comprising emodin.

Description

Emodin for the preparation of treatment of retinal ischemia or diseases, conditions or disorders associated with retinal ischemia use of the drug

The present invention relates to the use of emodin for preparing medicine for treating retinal ischemia. The present invention more particularly relates to the use of a composition comprising emodin for the manufacture of a medicament for the treatment and/or prevention of retinal ischemia and diseases, conditions or disorders associated with retinal ischemia.

Clinical relevance of retinal ischemia models

Retinal vascular occlusion (central retinal artery/vein occlusion and branch retinal artery/vein occlusion), glaucomatous optic neuropathy (glaucomatous optic neuropathy), proliferative diabetic retinopathy (DBR), and neovascular age-related macular Both neovascular age-related macular degeneration (AMD) and retinal dysplasia are diseases associated with retinal ischemia. Retinal ischemia can be detected when retinal thinning and/or visual field changes due to the death of internal retinal neurons are evidenced by optical coherence tomography (OCT). retinal ganglion cells (RGCs). Clinically, when b-wave changes in electroretinogram (ERG), retinal thinning as evidenced by optical coherence tomography (OCT) and/or due to intraretinal neurons (eg retinal ganglion cells ( retinal ganglion cells, RGCs)) die Retinal ischemia can be detected when visual field changes occur. These diseases affect millions of people worldwide, so the treatment of retinal ischemia is very important. A model involving the induction of retinal ischemia was therefore established, which model included increased intraocular pressure (IOP). Utilizing this approach to study novel therapeutic approaches related to various signaling pathways will be a useful approach to finding suitable anti-retinal ischemia drugs.

Exploration of β-catenin (β-catenin) and Vascular Endothelial Growth Factor (VEGF)

Studies have shown that under normoxia, the signaling pathway of β-catenin activates T cell factor 4 (TCF-4) and promotes cell proliferation. However, in the presence of hypoxia, β-catenin promotes the expression of hypoxia-inducible factor-1α (HIF-1α), which is known to subsequently increase vascular endothelial growth factor ( VEGF). This event in turn arrests the cell cycle to adapt to hypoxia. Furthermore, it has been reported that inhibition of the protein expression of β-catenin and/or vascular endothelial growth factor (VEGF) prevents ischemia-induced increases in vascular permeability that are associated with subsequent neovascularization, ocular Bleeding and/or cystoid macular edema.

Emodin

Emodin (6-methyl-1,3,8-trihydroxyanthraquinone; 6-methyl-1,3,8-trihydroxyanthraquinone) is a source of rhubarb, buckthorn and Japanese knotweed (Japanese knotweed). Emodin has been shown to have the potential to inhibit inflammation under various conditions. For example, emodin has been shown to reduce the severity of experimental disease models, including arthritis, liver damage, atherosclerosis, myocardial ischemia and cancer.

The present invention relates to a method for treating retinal ischemia or retinal ischemia in an individual in need thereof. A method of a disease, condition or disorder associated with membrane ischemia comprising administering to the individual an effective dose of emodin or a composition comprising emodin.

In the present invention, an animal model was used to study the protective effect of emodin on retinal ischemia. In addition, the present invention also studies how to regulate the amount of β-catenin when emodin treats retinal ischemia.

The present inventors have demonstrated that retinal ischemic damage due to increased intraocular pressure (IOP) can be alleviated by administration of emodin before and/or after ischemia. The above ischemia-induced changes were detected by electroretinography (ERG), histopathology (thickness of retinal layers stained with cresyl violet), fluorescent gold reverse immunolabeled retinal ganglion cells (RGC), and β-chain The protein (β-catenin) and the expression of vascular endothelial growth factor (VEGF) were monitored by methods such as measurement of protein expression. In cell viability assay (MTT assay), administration of 0.5 μM emodin before Oxygen Glucose Deprivation (OGD) (intravitreal equivalent injection of 20 μM emodin in rats) significantly attenuated OGD-induced Cell damage. In addition, in animal experiments, intravitreal injection of 20 μM emodin before ischemia significantly reduced the decrease in b-wave amplitude in electroretinogram (ERG) caused by retinal ischemia. The post-ischemic intravitreal injection group also significantly alleviated the decrease in b-wave amplitude. This protection was also present when using cresyl violet-stained retinal thickness and/or retrograde fluorescent gold immunolabeled RGC density. In addition, the present invention has demonstrated that the protein expression level of β-catenin/vascular endothelial growth factor (VEGF) is significantly increased after retinal ischemic injury. This elevation was significantly suppressed by preconditioning with emodin before ischemia. The above findings imply that emodin has a protective effect on retinal ischemia-damaged neurons such as retinal ganglion cells (RGCs) by down-regulating ischemia-induced up-regulation of β-catenin/vascular endothelial growth factor (VEGF). .

Under ischemic (hypoxic) conditions, up-regulated HIF-1α protein competes with TCF-4 for binding to β-catenin in cells, and once bound to HIF-1α, β-catenin will rapidly deactivate from co-activation of TCF-4 shifts its role to a role in stimulating HIF-1α-mediated transcription. Under ischemic conditions, increased HIF-1α-mediated transcription results in upregulation of downstream vascular endothelial growth factor (VEGF) with a range of possible sequelae, including macular edema and/or ocular hemorrhage. The present invention shows that emodin has an ameliorating effect on retinal ischemic damage, and at the same time dose-dependently and significantly (at 20 μM) ischemia-induced overexpression of β-catenin/vascular endothelial growth factor (VEGF) down. Thus, the protective mechanism of emodin appears to involve its inhibition of β-catenin/vascular endothelial growth factor (VEGF) protein expression, as well as a reduction in the degree of transcription and transcription mediated by HIF-1α co-activated by the aforementioned β-catenin proteins. A subsequent decrease in VEGF concentration. All these findings and the current protein analysis (Figure 5) strongly support the anti-ischemic and protective effects of emodin through inhibition of ischemia-induced β-catenin and vascular endothelial growth factor (VEGF) ) overexpression (Figure 5). This is of great clinical importance as it may point to a way to manage clinically proven complications from retinal ischemic disease such as macular edema. These include retinal vascular occlusion, proliferative diabetic retinopathy, neovascular age-related macular degeneration, and possible retinal developmental disorders (eg, familial exudative vitreoretinopathy (FEVR), Coat's disease, Persistent Hyperplastic Primary Vitreous (PHPV), Norrie disease or Retinopathy of Prematurity (ROP)).

Accordingly, the present invention provides a method of treating retinal ischemia or a disease, condition or disorder associated with retinal ischemia in an individual in need thereof, comprising administering to the individual an effective dose of emodin or a combination comprising emodin thing. The effective dose of emodin administered to the individual by the present invention can be 2 to 30 μM; in some embodiments, the effective dose of emodin administered to the individual can be 4 to 20 μM; in one embodiment, the effective dose of emodin administered to the individual is selected from 4 μM , 10 μM and 20 μM. In one embodiment of the present invention, the emodin is administered by intravitreal injection. In another embodiment of the present invention, the emodin is administered orally.

In one embodiment, the disease, condition or disorder associated with retinal ischemia comprises retinal vascular occlusion, glaucomatous optic neuropathy, proliferative diabetic retinopathy, neovascular age-related macular degeneration (AMD), familial exudative Vitreoretinopathy (FEVR), Coat's disease, Persistent Hyperplastic Primary Vitreous (PHPV), Norrie disease, or Retinopathy of Prematurity (ROP) ). In a preferred embodiment, the disease, condition, or disorder associated with retinal ischemia comprises familial exudative vitreoretinopathy, Korotkick's disease, residual proliferative primary vitreous disease and Norrie's disease and retinopathy of prematurity .

In one embodiment, the individual refers to a mammal, and in a more preferred embodiment, the individual refers to a human.

In one embodiment, the methods of the present invention further comprise administering to the individual a pharmaceutically acceptable adjuvant, vehicle or carrier.

In one embodiment, the composition is for the treatment of retinal ischemia or a disease associated with retinal ischemia by inhibiting the overexpression of β-catenin and vascular endothelial growth factor (VEGF) caused by retinal ischemia , condition or disease.

The present invention also provides a method of modulating β-catenin for the benefit of treating or lessening the severity of a disease, condition or disorder, comprising treating a person suffering from the disease, condition or disorder. An individual with the disorder is administered a therapeutically effective dose of emodin or a composition comprising emodin. In one embodiment, the effective dose of emodin administered to the individual of the present invention may be 2 to 30 μM; in another embodiment, the effective dose of emodin administered to the individual may be 4 to 20 μM; In a preferred embodiment, the effective dose of emodin administered to the individual is selected from the group consisting of 4 μM, 10 μM and 20 μM. In one embodiment of the invention, the emodin is administered to the individual by intravitreal injection. In another embodiment of the present invention, the emodin is administered orally to the individual.

In one embodiment, the individual refers to a mammal, and in a more preferred embodiment, the individual refers to a human.

In one embodiment, the methods of the present invention further comprise administering to the individual a pharmaceutically acceptable adjuvant, vehicle or carrier.

The present invention further provides a method of protecting cells in an individual in need thereof from damage caused by retinal ischemia, the method comprising administering to the individual an effective dose of emodin or a composition comprising emodin, Wherein the cell line is selected from bipolar cells (bipolar Cells), Müller cells (Müller Cells) and cholinergic amacrine cells. In one embodiment, the effective dose of emodin administered to the individual of the present invention may be 2 to 30 μM; in another embodiment, the effective dose of emodin administered to the individual may be 4 to 20 μM; In a preferred embodiment, the effective dose of emodin administered to the individual is selected from the group consisting of 4 μM, 10 μM and 20 μM. In one embodiment of the present invention, the emodin is administered by intravitreal injection. In another embodiment of the present invention, the emodin is administered orally.

In one embodiment, the composition protects cells in an individual in need thereof from retinal ischemia by inhibiting retinal ischemia-induced overexpression of β-catenin and vascular endothelial growth factor (VEGF). blood damage.

The present invention provides the use of a composition for the manufacture of a medicament for treating or preventing retinal ischemia or a disease, condition or disorder associated with retinal ischemia in an individual in need thereof, wherein the composition comprises emodin.

In one embodiment of the present invention, the effective dose of emodin may be 2 to 30 μM; in another embodiment, the effective dose of emodin may be 4 to 20 μM; in a preferred embodiment, The effective dose of the emodin is selected from 4 μM, 10 μM and 20 μM. In one embodiment of the present invention, the emodin is administered by intravitreal injection. In another embodiment of the present invention, the emodin is administered orally.

In the present invention, the clinical importance of emodin is that it can be used to treat retinal ischemic diseases or clearly associated complications (eg macular edema) due to retinal ischemia, namely central/branch retinal vessel occlusion, hyperplasia Diabetic retinopathy, neovascular age-related macular degeneration (AMD), and retinal developmental disorders such as familial exudative vitreoretinopathy (FEVR), Coat's disease, residual proliferative primary vitreous disease ( Persistent Hyperplastic Primary Vitreous (PHPV), Norrie disease or Retinopathy of Prematurity (ROP).

In one embodiment, the individual refers to a mammal, and in a more preferred embodiment, the individual refers to a human.

In one embodiment, the methods of the present invention further comprise administering to the individual a pharmaceutically acceptable adjuvant, vehicle or carrier.

The present invention further provides the use of a composition for the manufacture of a medicament for protecting cells in an individual in need thereof from damage caused by retinal ischemia, wherein the composition comprises emodin.

In one embodiment of the present invention, the cell line is selected from bipolar cells, Müller cells and cholinergic amacrine cells. In one embodiment of the present invention, the effective dose of emodin may be 2 to 30 μM; in another embodiment, the effective dose of emodin may be 4 to 20 μM; in a preferred embodiment, The effective dose of the emodin is selected from 4 μM, 10 μM and 20 μM. In one embodiment of the present invention, the emodin is administered by intravitreal injection. In another embodiment of the present invention, the emodin is administered orally.

In one embodiment, the medicament treats or prevents retinal ischemia in the individual in need thereof by inhibiting the excessive expression of β-catenin (β-catenin) and vascular endothelial growth factor (VEGF) caused by retinal ischemia or A disease, condition or disorder associated with retinal ischemia.

Abbreviations: ERG: electroretinogram; OCT: optical coherence tomography; RGC: retinal ganglion cells; IOP: intraocular pressure/intraocular pressure; HIOP: high intraocular pressure/high intraocular pressure; TCF-4: T-cytokine- 4(T-cell factor-4); HIF-1α: Hypoxia-inducible factor-1α (Hypoxia-inducible factor-1α); OGD: Oxygen glucose deprivation; MTT: 3-(4,5-dimethylthiazole-2) -yl)-2,5-diphenyltetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); I/R: ischemia plus reperfusion; Emo : Emodin; ARRIVE: Animal Research: Reporting of In Vivo Experiments; ILM: Internal limiting membrane; RPE: retinal pigment epithelium; INL: Inner nuclear layer ; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; PVDF: polyvinylidene fluoride; P: probability; VEGF: vascular endothelial growth factor.

FIG. 1 is a cell viability assay (MTT assay) to quantify the viability of cultured RGC-5 cell lines. The value of each group is the ratio of the survival rate of cultured RGC-5 cells to the survival rate of the control group, where the survival rate of the control group was set as 100%. The study included four groups, namely the control group (normal; RGC-5 cells were cultured in medium containing vehicle), the pre-OGD vehicle group (treated with vehicle for 1 hour before OGD), and the pre-OGD Emo 0.25 μM group (treated with 0.25 μM emodin for 1 hour before OGD) and Emo 0.5 μM group before OGD (treated with 0.5 μM emodin for 1 hour before OGD). *** indicates a significant difference between the normal control group and the OGD group ( P < 0.001). † Indicates a significant difference between the pre-OGD vehicle group and the pre-OGD Emo 0.5 μM group ( P =0.04). Results are mean±standard error (n=5~6). Abbreviations: RGC-5: retinal ganglion cell-5; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); OGD: oxyglucose deprivation; Emo: emodin.

Figure 2 shows the measurement results of electroretinogram (ERG) b waves. Figure 2a shows the amplitude of the b-wave of electroretinogram (ERG). After ischemia/reperfusion (I/R), compared with the retina of the sham-operated control group (Sham), the pre-intravitreal vehicle (Vehicle) group (Vehicle+I/R) was pre-administered with the retinal ischemia. The amplitude of the ERG b wave was substantially reduced; in a dose-dependent manner, Emodin (Emodin) was pre-administered in the vitreous (the doses were 4 μM, 10 μM and 20 μM, namely: Emo4+I/R, Emo10+I/ The reduction in ERG b-wave amplitude due to ischemia was improved in the R and Emo20+I/R) groups, but not in the vehicle group. The group (I/R+Emo20) injected with 20 μM of emodin intravitreal after ischemia also showed an anti-ischemic effect. Figure 2b is an analysis of b-wave ratios in electroretinogram (ERG) showing the efficacy of emodin pre- and post-ischemic administration on the ischemic retina. Compared to the ERG b-wave ratio in the sham control group (Sham=0.82±0.14: normalized to 1, n=7), the vehicle+I/R group showed a significantly lower b-wave ratio (***; P <0.001). ). Compared with the vehicle+I/R group (0.04±0.01, n=8), the pre-ischemic intravitreal injection dose-response emodin group (Emo4+I/R=0.08±0.07, n=8, Emo10 +I/R=0.64±0.28, n=4, and Emo20+I/R=0.99±0.18, n=4) significantly reduced ischemic injury (†††; P <0.001; Emo10+I/R) group and Emo20+I/R group). Intravitreal injection of 20 μM emodin after ischemia (I/R+Emo20=0.24±0.09, n=9) also had a significant anti-ischemic effect (†††; P < 0.001). Data are mean ± standard error of the mean (SEM) for the number of animals (n) shown in parentheses. Table 3 provides abbreviations for each group name.

Figure 3 is a calculation of retinal thickness stained with cresyl violet. Figures 3a to 3f are cresyl violet stained retinal sections with the same eccentricity. Microscopic images show the thickness (μm) of the whole retina (upper row) or inner retina (lower row) of different groups. Figures 3a, 3b, 3g and 3h show that the whole retina of the vehicle + I/R group was compared with the retinal thickness of the control group (sham control: whole thickness = 186.50 ± 1.43; inner layer thickness = 79.90 ± 2.06). or a substantial reduction in the thickness of the inner retina (whole thickness = 71.80 ± 1.08; inner thickness = 20.97 ± 0.85). Figures 3c to 3e, 3g and 3h show the dose-response of pre-ischemic intravitreal injection of emodin (the least effect at 4 μM, Emo4+I/R group: whole thickness=87.40±0.60, inner layer thickness=38.60±1.01; followed by 10 μM; maximal effect at 20 μM), and attenuated ischemia-induced reduction in whole retinal and inner retinal thickness in a significant manner (Emo10+I/R group: whole thickness=153.20±1.48, inner thickness=70.05±0.60; Emo20 +I/R group: overall thickness=170.10±0.10, inner layer thickness=70.65±2.06). Figures 3f, 3g and 3h show that intravitreal injection of 20 μM emodin after ischemia also significantly attenuated the reduction in the thickness of the whole retina and inner retinal layer caused by retinal ischemia (I/R+Emo20: whole thickness=125.45±1.68 , inner layer thickness=69.65±0.68). Figures 3g and 3h are quantitative analysis of whole retina or inner retinal thickness. *** or †††/†† indicates a significant difference ( P < 0.001 or P < 0.001/ P < 0.01) compared to the sham-operated control group (Sham) or the vehicle + I/R group. Abbreviations used in the figures are as follows: I/R: ischemia plus reperfusion; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer ; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar is 50 μm. Results are mean ± standard error. Table 4 provides abbreviations for each group name.

Figure 4 is a fluorogold reverse label. Microscopic images show the density of retinal ganglion cells (RGCs) in each group. Figures 4a and 4e show that in the sham-operated control group, the highest density of RGCs observed was 5323.53 ± 215.6/0.17 mm ² . Figures 4b and 4e indicate that the cell density was significantly reduced in the vehicle + I/R group. Figures 4c, 4d and 4e show that pre-ischemic intravitreal injection of emodin 10 μM and 20 μM (Emo10+I/R and Emo20+I/R) was dose-dependent and significantly increased cell density. Figure 4e shows quantitative analysis of retinal ganglion cell (RGC) density. *** or ††/††† indicates a significant difference ( P < 0.001 or P < 0.01/ P < 0.001) from the sham-operated control group or the vehicle + I/R group. Emodin was dose-dependent and significantly counteracted the reduction in retinal ganglion cell (RGC) density induced by retinal ischemia. Results are expressed as the mean ± standard error of the number of animals shown in parentheses. Scale bar is 50 μm. Table 5 provides abbreviations for each group name.

Figure 5 shows the detection and analysis of Western blotting method. Figure 5a shows the blot images of β-catenin, vascular endothelial growth factor (VEGF) and β-actin, lane 1 from the retina of the sham-operated control group (control group); lane 2 is ischemic retina pretreated with vehicle (vehicle+I/R); lanes 3 and 4 are from emodin (Emo20+I/R) at 10 μM (Emo10+I/R) and 20 μM, respectively. R) Retinal pre-treated and post-ischemia-reperfused. Figure 5b and Figure 5c are bar graphs of the ratio of β-catenin and vascular endothelial growth factor (VEGF) to house-keeping protein β-actin, respectively. The ratio of the sham-operated control group was normalized to 1, ***/* indicates a very significant difference ( P < 0.001) or a significant difference ( P < 0.05) compared with the sham-operated control group, † indicates a difference with vehicle + Compared with the I/R group, there was a significant difference ( P = 0.02/0.03). Significantly increased protein expression levels of β-catenin/vascular endothelial growth factor (VEGF) were observed following ischemic injury and pre-ischemic vehicle application compared to sham-operated controls (vehicle+I/R= 1.64±0.14/7.67±2.57). In contrast, administration of emodin before ischemia dose-dependently and significantly inhibited ischemia-induced increases in the protein expression of β-catenin ( P = 0.02/0.03 at 20 μM emodin ; Emo20+IR=1.00±0.19/1.23±0.44). Values are the mean ± standard error of the number of animals (n) indicated in parentheses. Abbreviations are listed as follows: I/R: ischemia plus reperfusion; Emo10+I/R: 10 μM emodin given before ischemia followed by I/R; Emo20+I/R: 20 μM Emodin given before ischemia flavin, followed by I/R. Abbreviations for each group name are provided in Table 6.

The following examples are non-limiting and represent only various aspects and features of the present invention.

Materials and Methods

cell experiment

oxyglucose deprivation

RGC-5 cells have been reported not to be transformed rat RGCs, but to mouse retinal neuronal precursor cells. OGD was established under ischemia-mimicking conditions by incubating RGC-5 cells at 37°C in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific Inc.) without glucose, i.e. 1% oxygen (using a Penguin Incubator ; Astec Company, Kukuoka, Japan measurement), 94% nitrogen and 5% carbon dioxide. studied various Experimental group (Table 1). These are cells treated with: (i) medium containing vehicle (control group), (ii) pre-OGD treatment with vehicle for 1 hour (OGD pre-vehicle group), (iii) with 0.25 μM 1 hour pre-OGD treatment with flavin (pre-OGD Emo 0.25 μM group), (iv) 1 hour pre-OGD treatment with 0.5 μM emodin (pre-OGD Emo 0.5 μM group). After 24 hours of OGD, the cell cultures were transferred to new DMEM for an additional day and then subjected to a cell viability assay (MTT assay) to assess cell viability.

Cell viability assay (MTT assay)

，從而增加暗紫色甲

的量，這與更大的細胞存活率有關。將MTT(0.5mg/mL；Sigma-Aldrich)添加到96孔盤中每孔100μL的細胞中，在37℃下放置3小時。還原MTT後，透過添加100μL DMSO溶解甲

。搖動後，使用ELISA酶標儀(Synergy H1 Multi-Mode Reader BioTek Instruments)在562nm波長下測量溶解的甲

的光密度(OD)。與未處理的對照組(100%)相比，將OD值的變化計算為細胞存活率。 Mitochondrial nicotinamide adenine dinucleotide phosphate-dependent oxidoreductase reduces MTT production of formazan

, thereby increasing the dark purple armor

amount, which was associated with greater cell viability. MTT (0.5 mg/mL; Sigma-Aldrich) was added to 100 [mu]L per well of cells in a 96-well plate and left at 37[deg.]C for 3 hours. After reducing MTT, dissolve formazan by adding 100 μL DMSO

. After shaking, the dissolved formazan was measured using an ELISA plate reader (Synergy H1 Multi-Mode Reader BioTek Instruments) at a wavelength of 562 nm.

optical density (OD). The change in OD value compared to the untreated control (100%) was calculated as cell viability.

The value of each group is the ratio of the survival rate of cultured RGC-5 cells to the survival rate of the control group, where the survival rate of the control group was set as 100%. The study included 4 groups including control (normal; RGC-5 cells, incubated in vehicle-containing medium), pre-OGD vehicle group (vehicle given 1 hour before OGD onset) , pre-OGD Emo 0.25μM group (0.25μM emodin administered 1 hour before OGD) and pre-OGD Emo 0.5μM group (0.5μM emodin administered 1 hour before OGD). *** indicates a very significant difference between the normal control group and the OGD group ( P < 0.001). † Indicates a significant difference between the pre-OGD vehicle group and the pre-OGD Emo 0.5 μM group ( P =0.04). Results are mean±standard deviation (n=5~6). Abbreviations: RGC-5: retinal ganglion cell 5; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4,5 -dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); OGD: oxyglucose deprivation; Emo: emodin.

Animal experiment

laboratory animal

The experimental animals used in the present invention are Wistar rats purchased from Taipei BioLasco Company. Upon arrival, Wistar rats were 6 weeks old and weighed 175 to 250 grams. The rats were housed in a large plastic rat cage (Shin Tak Instruments Co., Ltd.) in groups of less than six rats. Humidity and temperature were controlled within 40-60% and 21±2℃, respectively. Rats were randomly assigned to each experimental group. The number of rats used in the present invention is 120 (99 + 21, 99 is 60 + 16 + 23, as shown in Table 2), including animals that died during the following experiments (n =21): These occurred during retinal ischemia induction (n=9), ERG detection (n=4) and fluorescent gold reverse-labeled RGCs (n=8). Following ERG recordings, all surviving rats (n=99) were subjected to cresyl violet staining, fluorescent gold labeling and Western blotting. Surviving animals underwent sham surgery (Sham) procedure, vehicle pre-ischemia or emodin (Emo) pre/post-ischemia and were divided into the following groups: sham control group (n=19); vehicle + I/R group (n=20); Emo4+I/R (n=10); Emo10+I/R (n=20); Emo20+I/R (n=20) and I/R+Emo20 (n =10). Animal experimental procedures are based on the "Animal Research Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

Animal anesthesia and euthanasia

Wistar rats were anesthetized with an intraperitoneal injection of a combination of ketamine (Pfizer; 100 mg/kg) and xylazine (Sigma-Aldrich; 5 mg/kg) for analgesia and sedation. For ERG recordings, intracranial injections of fluorescent gold and intravitreal injections of specific agents were performed in Wistar rats. To minimize pain and to sacrifice Wistar rats humanely, each individual was finally injected intraperitoneally with at least 140 mg/kg of sodium pentobarbital (SCI Pharmtech).

drug administration

Emodin was purchased from Sigma-Aldrich (E7881; 90%; stored at 2-8°C; purchased from Frangula bark; St. Louis, Missouri, United States). Tables 3 to 6 describe the different drug administration procedures, namely electroretinography (ERG), fluorescent gold RGC labeling, cresyl violet retinal staining and western blotting. Vehicle or emodin (5 μL) was administered by intravitreal injection so that the agents could diffuse directly to the target. Emodin was dissolved in vehicle (DMSO:distilled water=1:3). The final concentrations administered were 0.25 μM or 0.5 μM according to an arbitrary definition of vitreous volume of 200 μM, which was obtained after 40-fold dilution from the stock solution, i.e., 10 μM or 20 μM. To test a wider range of dose responses, 4 μM emodin was therefore added in animal experiments. Vehicle+I/R groups and emodin+I/R groups (emodin 4 μM, 10 μM or 20 μM administered before ischemia) were performed according to the following experimental procedures; these were the initial pre-ischemic intravitreal injections (vehicle or flavin), retinal ischemia was induced 1 day later, and animals were sacrificed the following day for various postmortem procedures other than electroretinography (ERG). In the post-ischemic treatment group, in order to reduce the number of animals used, only a single emodin concentration of 20 [mu]M was used in the present invention. In addition, emodin was administered via intravitreal injection 1 day after initial retinal ischemia induction, and animals were sacrificed the following day for the various postmortem procedures described above.

Induce retinal ischemia

The IOP procedure used increased IOP and was designed to induce retinal ischemia in Wistar rats and to simulate ischemic retinal disease. The rat was first anesthetized and restrained in a stereotaxic frame, a 30G needle was attached to a raised saline bottle, and the needle was pierced into the Anterior chamber of rat eye. The injection pressure of saline in the bottle was controlled at 120 millimeters of mercury (mmHg), and this step was continued for 1 hour. Successful induction of retinal ischemia was confirmed by the observation of retinal whitening. Animals were placed on a heating pad to maintain their body temperature throughout the experiment. The sham procedure was performed with the pressure of the saline in the bottle maintained at 0 millimeters of mercury (mmHg).

Electroretinogram (ERG) measurement recording

Animals were anesthetized prior to flash ERG measurements. For sham-operated control group (Sham) or pre-ischemia treatment group (intravitreal injection of emodin or vehicle 1 day before ischemia; ie emodin+I/R group or vehicle+I/R group) , scintillation ERG responses were measured before sham surgery, ischemic procedures, or any dosing step (day 0), and one day after sham surgery or pre-ischemic ischemic procedures with intravitreal administration. In the post-ischemia treatment group, ERG recordings were collected before ischemia (day 0) and after ischemia (time points: 1 day after ischemia; 1 day after post-ischemia intravitreal injection of emodin). Eight hours before ERG measurements, dark adaptation was performed and pupil dilation was induced using 1% tropicamide and 2.5% phenylephrine. ERG recording equipment was obtained from Grass-Telefactor Corporation (AstroNova, QC, Canada) and included a stimulator (PS22), a regulated power supply (RPS107) and an amplifier (P511). A strobe light (0.5 Hz) as a stimulus source was placed 2 cm directly in front of the eyes of the rats. Continuous measurements (10 kHz) were made with 15 blinks every two seconds and the amplitude was calculated to obtain an average value. For comparisons between groups, the ratio of b-wave amplitudes in one eye (sham or ischemic injury and given specific agents) to b-wave amplitudes in untreated normal eyes was analyzed.

cresyl violet staining

After the aforementioned rats were sacrificed, the rats were intracardially perfused with physiological saline, and then the eyeballs were enucleated and fixed with 4% paraformaldehyde at 4°C for 1 day. It was then dehydrated with ethanol, embedded in paraffin (Tissue-Tek TEC 5; Sakura), and sectioned to a thickness of 5 μm, after which the sectioned samples were stained with cresyl violet and examined by light microscopy (Leica). All sectioned retinal samples were taken at the same magnification (Ilford Pan-F plus film, 50 ASA).

The thickness of retinal sections was determined by taking retinal samples at the same distance (1.5 mm from the optic disc). To assess the degree of damage caused by retinal ischemia, the present invention measures the thickness of the entire retina (from the inner limiting membrane (ILM) to the retinal pigment epithelium (RPE) layer) and the thickness of the inner retina (from the ILM to the inner nuclear layer (INL) )). All measurements were performed by experts who were unaware of the experimental conditions for sample handling.

Fluorescent gold reverse labeling of RGC

On the first day of the experiment, after the rats were anesthetized, a 2 cm opening was made in the skin of their head, and two circular lateral openings were made in the skull with a drill, and then a Hamilton microsyringe (Hamilton Microsyringe) intracranial injections of 2 μL of 0.5% fluorescent gold (Sigma-Aldrich) were performed at depths of 3.8 mm, 4.0 mm and 4.2 mm below the apex of the skull. One day after luciferase treatment, administer emodin, vehicle or perform a sham procedure (as described in the drug administration paragraph) prior to ischemia. Rats were sacrificed 3 days after fluorescent gold injection. After sacrifice, retinal tissue was carefully dissociated, incubated with 4% paraformaldehyde fixative, sectioned, and processed. Finally, sample analysis was performed by fluorescence microscopy. The RGC density in this experiment was defined as the total amount of RGCs divided by the entire retinal area, and then the average was calculated.

Western ink dot method

In this experiment, retinal samples were taken immediately after the rats were sacrificed, and then lysis buffer (mammalian protein extraction reagent; Hycell) was used to obtain denatured proteins, which were then sonicated and quantified to a protein sample equivalent of 30 μg/30 μl per well. Prepared protein samples were separated on 12% Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA) and further blotted onto a polyvinylidene fluoride (PVDF) membrane. The transfected PVDF membrane was incubated with blocking buffer (Blocking Buffer; 135 mM NaCl, 8.1 mM Na ₂ HPO ₄ , 1.5 mM KH ₂ PO ₄ , 2.7 mM KCl; pH 7.2) containing 5% skim milk at 4°C for 16 Hour. The PVDF membrane was next incubated for 1 hour at 25°C with various primary antibodies, including mouse anti-β-actin monoclonal antibody (ab6276; 1:5000; Abcam Inc., Cambridge, UK) ), rabbit anti-β-catenin monoclonal antibody (ab32572; 1:5000; Abcam Inc., Cambridge, UK) and rabbit anti-VEGF polyclonal antibody (A-20; 1:200; sc-152 ). Subsequently, the blots on this PVDF membrane were incubated with their appropriate secondary antibody, Horseradish Peroxidase (HRP)-Conjugated Goat Antibody, for 1 hour at 25°C. -Rabbit IgG; 111-035-003; 1:2000; Jackson ImmunoResearch) or horseradish peroxidase-conjugated goat anti-mouse IgG (Horseradish Peroxidase (HRP)-Conjugated Goat Anti-Mouse IgG; sc-2005; 1:2000; Santa Cruz Biotech). Finally, the PVDF membrane was developed using an enhanced chemiluminescence analysis system (HyCell). The PVDF membrane was then scanned using an imager (Amersham Imager; LifeSciences) and the amount of each protein was quantified by scanning densitometry.

Statistical analysis

The present invention uses an unpaired Student's t-test to compare the statistical differences between the two experimental groups. Probability (P) < 0.05 indicates a statistically significant difference. All results are presented as mean ± standard error.

result

cell experiment

Evaluation of the effect of emodin on ischemia-induced decrease in cell viability by MTT assay

The cultured cells are used to identify effective doses of the test agent emodin. The effect of emodin (0.25 [mu]M and 0.5 [mu]M) on cultured cells was assessed for 1 day using the MTT cell viability assay and a mimic model of cellular ischemia (ie, OGD). OGD is defined as culturing cells in glucose-free medium at 37°C under hypoxic conditions (ie, 1% _O2 ). The results of each experimental group relative to the control group (set at 100%; cells incubated in medium containing vehicle) are subsequently described (Figure 1 and Table 1). Cells were incubated in culture medium followed by vehicle (cell viability: 38.30 ± 2.51%) for one hour before OGD, emodin 0.25 μM for one hour before OGD (cell viability: 43.81 ± 3.75%) Emodin 0.5 μM was administered one hour before OGD (cell viability: 47.52±3.99%). Emodin treatment resulted in a dose-related and significant ( P =0.04; at 0.5 μM) attenuation of OGD-induced cellular damage compared to the vehicle-treated group.

Animal experiment

The effect of emodin on the reduction of ERG b-wave amplitude induced by ischemia

In the sham-operated control group, the b-wave amplitude of the ERG was measured as 0.87 mV/±1.00±0.00 mV (as shown in Fig. 2a, the amplitude ratio was 0.82±0.14 and normalized to 1 in Fig. 2b; Table 3; n= 7). After retinal ischemia, a significant decrease ( P < 0.001) in the b-wave amplitude/ratio of ERG was observed (vehicle + I/R = 0.03 mV as shown in Fig. 2a and vehicle + I/R = 0.03 mV as shown in Fig. 2b 0.04±0.02, n=8). Intravitreal injection of emodin before ischemia attenuated the significant decrease in the b-wave amplitude/ratio of ERG induced by ischemia (Fig. I/R=0.08±0.07, n=8; Emo10+I/R=0.58mV as shown in Fig. 2a, Emo10+I/R=0.64±0.28 as shown in Fig. 2bB, P < 0.001, n=4; as Figure 2a shows Emo20+I/R=0.63mV, and Figure 2b shows Emo20+I/R=0.99±0.18, P < 0.001, n=4). Interestingly, this experiment also demonstrated that intravitreal administration of emodin after ischemia also significantly ameliorated the above-mentioned ischemia-induced reduction in b-wave amplitude/ratio of ERG ( P < 0.001) (Figure 2a). I/R+Emo20=0.12mV, as shown in Figure 2b, I/R+Emo20=0.24±0.09, n=9). The b-wave of ERG is generally considered to be mainly used to reflect the light-induced activity of bipolar cells and Müller cells. Therefore, the above b-wave results of ERG suggest that emodin can protect bipolar cells and Müller cells from damage caused by retinal ischemia.

Effects of emodin on ischemia-induced retinal thickness reduction in cresyl violet staining

As shown in Figure 3 and Table 4, retinal thickness (μm) in the sham-operated control group (n=10) was measured as: the whole retinal thickness was 186.50±1.43 μm or the inner retinal thickness was 79.90±2.06 μm. After induction of retinal ischemia, a significant loss of whole retinal thickness and inner retinal thickness was observed (vehicle+I/R: whole retinal thickness=71.80±1.08 μm; inner retinal thickness=20.97±0.85 μm; P <0.001 ). However, intravitreal injection of emodin (4, 10 or 20 μM) prior to ischemia was dose-dependent (n=10, Emo4+I/R: whole retinal thickness = 87.40±0.60 μm, inner retinal thickness =38.60±1.01μm; n=10, Emo10+I/R: whole retinal thickness=153.20±1.48μm, inner retinal thickness=70.05±0.60μm; n=10, Emo20+I/R: whole retinal thickness=170.10 ±0.10 μm; inner retinal thickness = 70.65 ± 2.06 μm) and significantly alleviated retinal thickness reduction due to retinal ischemia and reperfusion (4 μM emodin: P <0.01; 10 or 20 μM emodin: P < 0.001). In addition, intravitreal injection of emodin after ischemia (n=10; I/R+Emo20: whole retinal thickness=125.45±1.68 μm, inner retinal thickness=69.65±0.68 μm) was associated with ischemia-related There was also a significant anti-ischemic effect in terms of retinal thickness loss ( P < 0.001).

Effects of emodin in fluorescent gold reverse labeling on ischemia-induced reduction in RGC density

As shown in Figure 4a, Figure 4e and Table 5, the evaluation of RGC immunolabeling confirmed that the cell density of RGCs after sham operation was 5323.53 ± 215.6/0.17 ^mm2 (sham operation control group; n=4). Intravitreal administration of vehicle prior to ischemia observed a very significant decrease in the cell density of RGCs in the ischemic retina ( P < 0.001) (n=4; vehicle+I/R=2069.12±212.82 mm ² ; Figure 4b, Figure 4e). In addition, administration of 10 μM or 20 μM of emodin before ischemia revealed a dose-dependent and significant increase in cell density (n=4, P < 0.01 at 10 μM, Emol0+I/R=3345.59±206.80 mm ² , Fig. 4c and Fig. 4e; n=4, P < 0.001 at 20 μM, Emo20+I/R=4623.53±179.48 mm ² , Fig. 4d and Fig. 4e).

Effects of emodin on ischemia-induced changes in the ratio of β-catenin/vascular endothelial growth factor (VEGF) expression to β-actin expression

Western blot analysis was used to study the therapeutic mechanism of emodin in improving ischemic injury. With reference to the immunoblot images (Fig. 5a and Table 6), compared with the intravitreal administration of vehicle (vehicle+I/R) before ischemia, 10 μM or 20 μM of emodin (Emo10+I/R) was administered before ischemia. R or Emo20+I/R) has a dose-dependent inhibitory effect on ischemia-induced β-catenin (β-catenin)/vascular endothelial growth factor (VEGF) overexpression. In quantitative analysis (Fig. 5b, Fig. 5c and Table 6), β-catenin (β-catenin)/vascular endothelial growth factor (VEGF):β-actin ( The β-actin) ratio was normalized to 1 to calculate the effect of emodin on the protein expression of β-catenin (β-catenin)/vascular endothelial growth factor (VEGF). After ischemic injury, emodin was observed to contribute to the β-chain elicitation of ischemic and pre-ischemic intravitreal administration of vehicle (vehicle+I/R=1.64±0.14/7.67±2.57; n=6). Protein (β-catenin)/vascular endothelial growth factor (VEGF) was significantly up-regulated ( P < 0.001) dose-responsive (weak effect when given 10 μM emodin, Emo10+I/R=1.18±0.24/3.99±2.86 , n=6) and significant ( P =0.02/0.03, P =0.02, Emo20+I/R=1.00±0.19, n=6) inhibition when 20 μM emodin was administered. Results shown in parentheses follow the beta-catenin/vascular endothelial growth factor (VEGF) format.

Those skilled in the art can easily understand the objects to be achieved by the present invention, and obtain the objects and technical advantages mentioned therein from the present invention. The methods and uses described herein represent preferred embodiments only, are exemplary, and are not intended to limit the scope of the invention. Any modifications and other uses conceived by those skilled in the art are included in the spirit of the present invention, and are defined by the scope of protection claimed by the patent application scope of the present invention.

It will be apparent to those skilled in the art that various substitutions and modifications of the invention disclosed herein can be made by those skilled in the art without departing from the scope and spirit of the invention.

The present invention may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. The terms used in the present invention are only used as descriptive terms and are not restrictive, and when these terms are used, they are not intended to exclude any equivalents of the technical features or parts thereof shown and described, but it should be recognized that in all Various modifications are possible within the scope of the claimed invention. Therefore, it should be understood that although the present invention has been specifically disclosed by the best or preferred embodiment and optional technical features, those skilled in the art will still Modifications and variations can be made with respect to the concepts disclosed herein, and such modifications and variations should be considered within the scope of the invention as defined by the appended claims.

Claims (4)

Use of a composition for the manufacture of a medicament for the treatment or prevention of retinal ischemia or a disease, condition or disorder associated with retinal ischemia in an individual in need thereof, wherein the composition comprises emodin, wherein the retinal ischemia The disease, condition or disorder or associated with retinal ischemia is retinal vascular occlusion, glaucomatous optic neuropathy, neovascular age-related macular degeneration (AMD), familial exudative Vitreoretinopathy (FEVR), Coat's disease, Persistent Hyperplastic Primary Vitreous (PHPV), Norrie disease, or Retinopathy of Prematurity (ROP) ), wherein the emodin is to treat the retinal ischemia or a disease, condition or disorder associated with retinal ischemia by inhibiting the overexpression of beta - catenin, wherein the composition is injected intravitreally The subject is administered with emodin in an amount of 4 to 20 μM in the composition. The use as claimed in claim 1, wherein the individual refers to a mammal. The use according to claim 1, wherein the individual is a human. The use of claim 1, wherein the composition further comprises a pharmaceutically acceptable adjuvant, vehicle, or carrier.

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