Low-Dose Trans-Resveratrol Ameliorates Diabetes-Induced Retinal Ganglion Cell Degeneration via TyrRS/c-Jun Pathway

Abstract

Purpose: The purpose of this study was to investigate the protective effect of low-dose trans-resveratrol (trans-RSV) on diabetes-induced retinal ganglion cell (RGC) degeneration and its possible mechanism.

Methods: A streptozotocin-induced diabetic mouse model was established and treated with or without trans-RSV intragastric administration (10 mg/kg body weight/day) for 12 weeks. Oscillatory potentials (Ops) of the dark-adapted electroretinogram (ERG) were recorded. The number of RGCs was detected by Tuj1 and TUNEL staining. The apoptosis markers in the retina were analyzed by Western blot. The cross sections of optic nerves were observed by transmission electron microscopy. In addition, mouse neuroblastoma N2a cells were injured by high-glucose (HG) treatment. Cell viability and apoptosis were measured with or without low-dose trans-RSV treatment. The intracellular localization of tyrosyl transfer-RNA synthetase (TyrRS) was observed in both mouse retinas and N2a cells. The effects of low-dose trans-RSV on the binding of TyrRS to the transcription factor c-Jun and the binding of c-Jun to pro-apoptotic genes were analyzed by co-IP and ChIP assays in HEK 293 cells.

Results: Trans-RSV relieved electrophysiological injury of retinas and inhibited RGC apoptosis in diabetic mice. It also protected N2a cells from HG-induced apoptosis. Additionally, it promoted TyrRS nuclear translocation in both diabetic mouse retinas and HG-treated N2a cells. Trans-RSV promoted TyrRS binding to c-Jun, inhibited the phosphorylation of Ser-63 of c-Jun, and downregulated pro-apoptotic gene transcription.

Conclusions: Low-dose trans-RSV can ameliorate diabetes-induced RGC degeneration via the TyrRS/c-Jun pathway. It can promote TyrRS nuclear translocation and bind to c-Jun, downregulating c-Jun phosphorylation and downstream pro-apoptotic genes.

Although diabetic retinopathy (DR) has long been considered a microvascular abnormality, increasing evidence suggests that the diabetic retina suffers from neuron loss and gliacyte proliferation in a manner similar to other neurodegenerative diseases, which is termed diabetic retinal neurodegeneration (DRN).¹ Previous clinical studies have reported functional abnormalities of the retina and the progressive loss of the nerve fiber layer and the ganglion cell/inner plexiform in patients with diabetes.^2,3 Laboratory research has demonstrated the apoptosis of retinal ganglion cells (RGCs) in streptozocin (STZ)-induced diabetic mice and Ins2^+/− diabetic mice, and investigated the mechanisms and therapeutic strategies (e.g. IL-17A knockout and somatostatin administration) of RGC injury in DRN.^4,5 Much remains unknown about the loss of RGCs in diabetes. Recently, tyrosine transfer-RNA synthetase (TyrRS), a member of the aminoacyl-tRNA synthetase (AARS) family, was reported to exert a neuroprotective effect on Alzheimer's disease (AD).⁶ High expression of TyrRS significantly attenuated oxidative DNA damage in primary rat cortical neurons.⁶ Moreover, TyrRS gene mutation was associated with the occurrence of the peripheral nerve degeneration diseases Charcot-Marie-Tooth and Leber hereditary optic neuropathy.^7–10 Both of these diseases cause optic atrophy and RGC death. TyrRS nuclear translocation has been considered a trigger of the stress response and plays a protective role in oxidative stress, serum starvation, heat shock, tunicamycin stimulation, and a general “stand-alone” stress response.^11–13 Acetylation modification of TyrRS is one way to promote its nuclear translocation and activate its antioxidant effects.¹³ Resveratrol (RSV) stimulation is another way that causes TyrRS nuclear translocation.¹² The phenolic rings of trans-resveratrol (trans-RSV) and tyrosine have similar spatial structures and can fit into the active site pocket of TyrRS, causing TyrRS nuclear translocation.¹² 

RSV exists in a variety of plants and is classified as cis- or trans-RSV according to the position of the two benzene rings binding to the carbon‒carbon double bond. There are some differences between cis- and trans-RSV in their vasodilatory, contractile, and anti-proliferative effects.^14,15 Unlike trans-RSV, cis-RSV is the isomer produced under ultraviolet light stimulation and is photosensitive and easily decomposed. The trans-RSV is more stable and currently available commercially.¹⁶ The study of trans-RSV may be instructive in practical applications. Due to its rapid metabolism and related low bioavailability, the application of trans-RSV has long been disputed and suspected.¹⁷ To determine the actual concentration of trans-RSV in human eyes, we analyzed vitreous humor samples from patients who underwent vitrectomy after trans-RSV oral ingestion.¹⁸ The mean concentration of the metabolites of trans-RSV (resveratrol-3-O-sulfate) was 62.95 ± 41.97 nmol/L, which was far below the doses used in previous experiments.¹⁸ The effect of low-dose trans-RSV remains to be investigated, and promoting TyrRS nuclear translocation may be its potential mechanism. 

To investigate the downstream pathway of TyrRS nuclear translocation, c-Jun, a prominent member of the activator protein-1 (AP-1) family, was identified as a potential target. c-Jun is involved in the regulation of apoptosis and other cell biological processes.¹⁹ The phosphorylation of Ser-63 and Ser-73 in the NH2-terminal transactivation domain substantially enhances its transcriptional activity.²⁰ The phosphorylation of c-Jun leads to the upregulation of a variety of pro-apoptotic genes, including BIM, HRK, FAS L, and p53.^21,22 Phosphorylation of Ser-63 is present in neurofibrillary tangles in human AD brains.²³ Dephosphorylation mutation of Ser-63 and Ser-73 can result in resistance to trophic factor deprivation and DNA damage in the mouse brain.²⁰ Similarly, in SH-Sy5y neuroblastoma cells, dephosphorylation mutation of Ser-63 can inhibit nitric oxide-induced caspase-3 activity and cell apoptosis.²² The phosphorylation of c-Jun has been considered to be regulated by c-Jun N-terminal kinase (JNK).²⁴ The link between c-Jun phosphorylation and TyrRS nuclear translocation remains to be established. 

In this study, we confirmed the protective effect of low-dose trans-RSV on diabetes-induced RGC degeneration and observed TyrRS nuclear translocation induced by trans-RSV. We detected the binding of exogenous or endogenous TyrRS to c-Jun and then assessed the effect of low-dose trans-RSV on the phosphorylation of Ser73 and Ser63 of c-Jun and the expression of associated pro-apoptotic genes. 

Methods

Human Sample Collection

Donor eyes obtained from the Eye Bank, Wuhan Red Cross Medical Center, Hubei, China, were evaluated. The donors included four subjects with diabetes (male patients, aged 76.50 ± 9.04 years old with 11.75 ± 4.65 years duration of diabetes) and four without diabetes (male patients, aged 82.25 ± 12.00 years old). The posterior part of the eyeballs was fixed in eye fixing solution (68% alcohol, 10% formalin, and 5% acetic acid) for 1 day at room temperature and then paraffin embedded and sectioned. The retinal samples of the contralateral eyeballs were quickly isolated on ice for Western blot analysis. The study protocols were approved by the ethics committee of Tongji Hospital, and all of the subjects provided prior written informed consent in accordance with the tenets of the Declaration of Helsinki. 

Animals

Male C57BL6 mice aged 8 weeks were obtained from Gem Pharmatech (Nanjing, China) and kept at Tongji Hospital Animal Center (Wuhan, China). Mice were kept in an animal house under a 12-hour light-dark cycle with food and water ad libitum. Four sets of mice were created: a control group, a control with trans-RSV treated group (RSV), an STZ-induced diabetes mellitus (DM) group, and a DM + RSV group. Twenty mice were included in each group. DM was induced by intraperitoneal injection of STZ (50 mg/kg; Sigma‒Aldrich, St. Louis, MO, USA) for 5 days. After 3 days, mice with blood glucose ≥16.7 mmol/L were considered to be diabetic. Trans-RSV (Sigma‒Aldrich) was administered by gavage at 10 mg/kg body weight/day for 12 weeks. Trans-RSV powder was stored away from light. An appropriate volume of suspension was freshly configured with PBS buffer for each use. After electroretinogram tests, the eyeballs were removed, paraffin embedded, and sectioned or separated on ice. All animal experiments were approved by the Institutional Animal Research Committee of Tongji Medical Center and were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA). 

Cell Culture

Mouse primary retinal cells were isolated from C57BL6 mice of postnatal day 5 according to the reported methods.²⁵ A single cell suspension of the retinal tissue was prepared by incubating retinas in Dulbecco's modified Eagle's medium/Nutrient Ham's Mixture F-12 (DMEM/F12; Gibco, Grand Island, NY, USA) supplemented with 18 units/mL papain solution, 0.3 g/mL L-cysteine, and 0.5 g/mL fetal bovine serum albumin for 30 minutes at 37°C and further mechanically dissociated with a burned-tip glass Pasteur pipette. Single cell suspension was centrifuged and resuspended in Neurobasal A medium (Gibco) supplemented with 2% vol/vol B27 (Gibco), 1% vol/vol N2 (Gibco), 100 U/mL penicillin G, and 100 mg/mL streptomycin sulfate (Servicebio, Wuhan, China), in a humidified incubator with 5% CO₂ and 37°C. Mouse primary retinal cell injury was induced by high-glucose (25 mM, high-glucose [HG], 72 hours) treatment. Then trans-RSV was added to a concentration of 100 nM for 24 hours. 

N2a cells were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology (ZQ0207; Shanghai, China) and cultured in DMEM (Gibco) supplemented with 10% vol/vol fetal bovine serum (Gibco), 100 U/mL penicillin G, and 100 mg/mL streptomycin sulfate (Servicebio) in a humidified incubator with 5% CO₂ and 37°C. Cells were divided into four groups: control, RSV, HG, and HG + RSV groups. When the cell density reached approximately 50% to 60%, the medium was changed so that the cell culture glucose concentration was 5 mM (control) or 25 mM (HG). Trans-RSV was added to bring the concentration to 10 nM unless otherwise stated. After 24 hours of incubation, the cells were fixed or lysed. 

HEK293 cells (ZQ0034; Shanghai Zhong Qiao Xin Zhou Biotechnology) were cultured in HG DMEM (Gibco) at a glucose concentration of 25 mM (HG) supplemented with 10% volume/volume fetal bovine serum (Gibco), 100 U/mL penicillin G, and 100 mg/mL streptomycin sulfate (Servicebio). The cells were incubated in a humidified incubator with 5% CO₂ at 37°C. When the cell density reached approximately 50% to 60%, the medium was replaced, and trans-RSV was added to a concentration of 100 nM. After 24 hours, the cells were collected for the next experiment. 

Electroretinogram

The electroretinogram (ERG) recorder (RetiMINER; IRC, Chongqing, China) was provided by the Eye Institute of Wuhan University. After at least 12 hours of dark adjustment, the mice were anesthetized by intraperitoneal injection of 5% chloral hydrate. Medolite eye drops were used to dilate the pupils. Then, dark-adapted ERGs (0.001, 0.003, 0.001, 0.003, 0.1, 0.3, 1.0, and 3.0 cd.s/m² flashes stimuli) were recorded according to the RetiMINER operating guide. Oscillatory potentials (OPs) were recorded at 3.0 cd.s/m² flash stimuli. 

Immunofluorescence Assay

Cell slides were fixed with 4% paraformaldehyde for 15 minutes after treatment. Paraffin-embedded tissue slides were routinely deparaffinized and hydrated and then immersed in citric acid/sodium citrate buffer for 7 minutes at high temperature and high pressure. Cell and tissue slides were blocked with 10% donkey serum in 0.3% Triton X-100 for 60 minutes at room temperature. Slides were incubated in diluent primary antibody overnight at 4°C, washed with PBS 3 times, and incubated in diluent containing Alexa Fluor 488/594-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 1 hour at room temperature. After DAPI staining for 5 minutes and another 3 washes with PBS, the slides were covered in AntiFade Mounting Medium (MCE) and then observed under a fluorescence microscope (Olympus BX53) or a confocal microscope (C2; Nikon, Tokyo, Japan). The primary antibodies used are listed in Supplementary Table S1. 

TdT-Mediated dUTP Nick-End Labeling Staining

The One Step TdT-mediated dUTP Nick-End Labeling (TUNEL) Apoptosis Assay Kit (MA0223; Meilun, Dalian, China) was used according to the manufacturer's instructions. Paraffin-embedded tissue slides were deparaffinized and hydrated, and 20 µg/mL proteinase K was used to permeabilize the tissues for 30 minutes at 37°C. N2a cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature. Proteinase K (20 µg/mL) was used to permeabilize the cells for 20 minutes at 37°C. The slides of both were incubated for 60 minutes at 37°C, protected from light, and then observed under a fluorescence microscope (488 nm; Olympus BX53). 

Fluoro-Jade C Staining

According to the instructions of Fluoro-Jade C RTD Stain Reagent (TR-100; Biosensis, Thebarton, Australia), paraffin-embedded tissue slides were routinely deparaffinized and hydrated and sequentially soaked in the staining solution. The slides were dried for at least 5 minutes, protected from light, and then cleared by brief immersion in xylene. After being covered with DPX, the slides were observed under a fluorescence microscope (488 nm; Olympus BX53). 

Transmission Electron Microscopy

The optic nerve tissues were collected at a position 1 mm from the posterior wall of the eyeball and cut into small segments of approximately 3 to 4 mm. Then, the tissues were fixed in 2% glutaraldehyde for 24 hours at 4°C. The following steps were performed according to a previous report.²⁶ The sections were observed with a Tecnai G2 20 TWIN microscope (FEI Company, Hillsboro, OR, USA). 

Western Blot

The retina and cell proteins for Western blot were performed as previously described.²⁷ All the above samples were separated by SDS‒PAGE. The antibodies used are listed in Supplementary Table S1. The grayscale values of the bands were evaluated by ImageJ software (National Institutes of Health) and normalized to ACTB or H3. 

Single-Cell Gel Electrophoresis (Comet Assay)

Referring to the reported methods,²⁸ approximately 10⁵ cells were resuspended in PBS, then thoroughly mixed with low melting point agarose and sandwiched between normal melting point agarose on frosted slides. The slides were stained with 20 µg/mL ethidium bromide staining solution (Sigma‒Aldrich) and observed under a fluorescence microscope (594 nm; Olympus BX53). Images were analyzed by the CaspLab - Comet Assay Software Project (CASP). 

Cell Counting Kit-8 Assay

N2a cells were seeded into 96-well plates at a density of approximately 5000 cells/well. Cells were divided into four groups and treated in a manner similar to the previous description, and another group treated with different RSV concentrations was set up under HG conditions. The following steps were performed according to the instructions of the CCK8 kit (C0037; Beyotime, Shanghai, China). The experiment was independently repeated four times. 

FLAG-Tagged TyrRS Plasmid Construction and Cell Transfection

FLAG-tagged TyrRS plasmid was purchased from Sino Biological (HG15135-NFl; Beijing, China) and transfected into HEK293 cells with LipoFectMax 3000 Transfection Reagent (FP318; ABP Biosciences, Wuhan, China). 

Co-Immunoprecipitation

The protein extraction and subsequent processing of HEK293 cells for co-immunoprecipitation (co-IP) were performed as described in previously reported methods.²⁹ Magnetic beads were purchased from MCE (Shanghai, China). The samples of binding protein were analyzed by Western blot. The antibodies used are listed in Supplementary Table S1. The grayscale values of the bands were evaluated by ImageJ software (National Institutes of Health) and normalized to input or ACTB. 

Chromatin Immunoprecipitation Assay and Quantitative Real-Time Polymerase Chain Reaction

Freshly collected HEK293 cells were processed according to the chromatin immunoprecipitation assay (ChIP) kit (P2078; Beyotime) instructions. The obtained DNA was purified by a DNA Purification kit (D0033; Beyotime). RNA from HEK293 cells was freshly extracted through the TRIzol/chloroform method and then processed according to the instructions of HiScript II Q Select RT SuperMix for qPCR (Vazyme, Nanjing, China). After dilution, 2 µL DNA samples were added to a volume of 10 µL containing 5 µL of 2 × SYBR Green master mix and 3 µL of each primer. ACTB was used as the housekeeping gene to normalize the expression levels of the BIM, FAS L, HRK, and p53 genes. The primers used were as follows: for BIM, F: 5′-ATTACCAAGCAGCCGAAGAC-3′ and R: 5′-TCCGCAAAGAACCTGTCAAT-3′; for FAS L, F: 5′-GCCTGTGTCTCCTTGTGATG-3′ and R: 5′-TGGACTTGCCTGTTAAATGGG-3′; for HRK, F: 5′-GGCAGGCGGAACTTGTAGGAAC-3′ and R: 5′-TCCAGGCGCTGTCTTTACTCTCC-3′; p53, F: 5′-CACTAAGCGAGCACTG-3′ and R: 5′-GGAGGTAGACTGACCC-3′; and ACTB, F: 5′-TTCTACAATGAGCTGCGTGTG-3′ and R: 5′-GGGGTGTTGAAGGTCTCAAA-3′. The cycling conditions were as follows: 5 minutes at 95°C, 40 cycles for 10 seconds at 95°C, and 30 seconds at 60°C. Each reaction was performed in triplicate. At the end of each cycle, the fluorescence signal of SYBR Green was measured. 

Statistical Analysis

All experiments were repeated three or more times. The data are presented as the mean ± standard deviation. All groups were normalized to the control group unless otherwise stated, and statistical analysis was carried out with unpaired Student's t tests, ANOVAs and Shapiro-Wilk tests, using Prism software (GraphPad, San Diego, CA, USA). P values < 0.05 were considered significant. 

Results

The Protective Effects of Low-Dose Trans-RSV on Diabetes-Induced RGC Degeneration

To confirm the protective effects of trans-RSV on diabetes-induced RGC degeneration, a mouse model was established by STZ intraperitoneal injection and trans-RSV intragastric administration (Supplementary Fig. S1A). No statistically significant difference was found in body weight (Supplementary Fig. S1B) or blood glucose (Supplementary Fig. S1C) between the DM and the DM + RSV groups. As OPs of ERG arise from the neural activity of the inner retina, it was performed to represent the electrophysiological function of RGCs. The amplitude of OPs in diabetic mice was obviously lower than that in control mice, whereas trans-RSV treatment alleviated this impairment (Fig. 1A). This trend was also observed in the a and b waves of ERG, which arise from the neural activity of the outer and middle retina (Supplementary Fig. S2). These results suggested that trans-RSV may have a protective effect on all retinal layers. Meanwhile, trans-RSV significantly increased the density of retinal ganglion cells (Fig. 1B) and reduced the number of TUNEL-positive cells in the ganglion cell layer compared to the DM group (Fig. 1C). Fluoro-Jade C staining, which labeled denatured neurons, showed relieved neurologic impairment after trans-RSV treatment (Fig. 1D). Synaptophysin has been regarded to reflect the density of synapses. Trans-RSV treatment increased the level of synaptophysin in diabetic mouse retina (Figs. 1E, 1G). Scanning electron microscopy images showed that the myelin sheath of the normal optic nerve was complete, clear and closely arranged (Fig. 1F). In the DM group, the inner cell body of the myelin sheath was atrophic, the organelles were swollen, and some myelin sheath was disintegrated or thin, whereas the structure of the inner cell body was comparatively complete in the DM + RSV group. Western blot analysis of whole retinal proteins also showed significant reductions in the apoptosis markers cleaved-PARP and cleaved-caspase-3 in the DM + RSV group compared to the DM group, and the level of the ganglion cell marker Thy-1 were increased in the DM + RSV group compared to the DM group (Fig. 1H). None of the above results showed significant differences between the control and control + RSV groups, suggesting that oral trans-RSV did not cause damage to RGCs. 

Figure 1.

 

Low-dose trans-RSV reduced diabetes-induced neurotoxicity in the mouse retina. Dark-adapted electroretinogram (ERG) images of each group (n = 6). (A) Oscillatory potentials (OPs) were recorded at 3.0 cd.s/m2 flash stimuli. The 0 indicates the baseline; N1, N2, and N3 indicates the first, second, and third troughs; and P1, P2, and P3 indicates the first, second, and third peaks. Bar graph of OP amplitudes (subtracting the corresponding valley value from the peak value); the sum on the right. (B) Immunofluorescence images of Tuj1 (red)-labeled RGCs of each group (n = 8). The number of RGCs in the DM group was approximately 64% of that in the control group, whereas the number increased to 83% of that in the control after RSV treatment. (C) TdT-mediated dUTP nick-end labeling (TUNEL) staining (green) labeled apoptotic cells (yellow arrow) and (D) Fluoro-Jade C staining (green) labeled degenerated cells in the retinal ganglion cell layer (n = 8). (E) Immunofluorescence images of synaptophysin (green) (n = 8). (F) Transmission electron microscopy images of a cross section of the mouse optic nerve (n = 4). (G, H) Western blot of panretinal protein showed increased expression levels of synaptophysin and the RGC marker Thy-1 and decreased expression levels of the apoptosis markers cleaved PARP and cleaved caspase-3 in the DM + RSV group compared to the DM group (n = 8). *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001; ns, no significant difference.

The Protective Effects of Low-Dose Trans-RSV on HG-Induced N2a Cell Apoptosis

To determine the direct effect of low-dose RSV on RGCs, mouse primary retinal cells were isolated according to reported methods,²⁸ and injury was induced by HG (25 mM, HG, 72 hours) treatment (Supplementary Fig. S3). After 24 hours of stimulation with RSV (100 nM), an increased number of Tuj1-positive cells and an increased length of nerve fibers were observed. Then, HG (25 mM)-treated mouse neuro-2a (N2a) cells were used to simulate retinal ganglion cells in the diabetic state. The polygonal shape of N2a cells gradually disappeared, and round, floating cells appeared in the HG group, whereas more cells retained their shape in the HG + RSV group (Fig. 2A). Low-dose trans-RSV treatment reduced the number of TUNEL-positive cells compared to the HG group (Fig. 2B). To detect the degree of DNA damage, a comet assay was performed (Fig. 2C). Compared with the HG group, low-dose RSV significantly reduced the percentage of DNA in the tail, the tail length, and the tail moment. The viability of N2a cells decreased to approximately 71% of the control, whereas in the HG + RSV group, it increased to approximately 84% of the control (Fig. 2D). Notably, the protective effect of trans-RSV on N2a cells did not appear to follow a dose-effect relationship, as the cell viability decreased with increasing trans-RSV concentration when the concentration was above 1 µM. The levels of the apoptosis markers cleaved-PARP and cleaved-caspase 3 were also decreased in the HG + RSV group compared to the HG group (Fig. 2E). These results suggested that low-dose trans-RSV alleviated HG-induced apoptosis in N2a cells induced by high glucose. 

Figure 2.

 

Low-dose trans-RSV alleviated the damage to N2a cells induced by high glucose. (A) Images of N2a cells under a white-light microscope. (B) TUNEL staining (green) labeled apoptotic cells. (C) Images of single-cell gel electrophoresis (comet assay) and bar graph of the percentage of DNA in the tail, the tail length, and the tail moment, analyzed by CaspLab - Comet Assay Software Project (CASP). (D) Cell viability of N2a cells was measured by CCK8 assay. (E) Western blot of N2a cells demonstrated decreased levels of the apoptosis markers cleaved PARP and cleaved caspase-3 in the HG + RSV group compared to the HG group. All above experiments were repeated six times. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001; ns, no significant difference.

Low-Dose Trans-RSV Promoted TyrRS Nuclear Translocation In Vitro and In Vivo

The colocalization of TyrRS with the nucleus was observed in N2a cells from both the RSV and HG + RSV groups, whereas the fluorescence of TyrRS in the control and the HG groups was mainly found in the cytoplasm (Fig. 3A). This result suggested that the TyrRS nuclear translocation induced by low-dose trans-RSV is not affected by HG conditions. Western blot analysis of cytoplasmic and nuclear proteins provided consistent results, with increased TyrRS expression in the nucleus following trans-RSV treatment (Fig. 3B). In the mouse retina, TyrRS nuclear translocation was also observed in the ganglion cell layers of the RSV and the DM + RSV groups, marked with yellow arrows (Fig. 3C). In the human retina, TyrRS expressed in the ganglion cell layer (GCL) and TyrRS level decreased in the DM group (Fig. 3D). 

Figure 3.

 

Low-dose trans-RSV promoted TyrRS nuclear translocation in vitro and in vivo. (A) The subcellular localization of TyrRS (red) in N2a cells was observed by confocal microscopy. (B) Western blot of isolated cytoplasm and nuclear proteins from N2a cells. The experiments of A and B were repeated six times. (C) Localization of TyrRS (green) in the mouse retina was observed by confocal microscopy (yellow arrow) (n = 8). (D) Immunofluorescence images of TyrRS (red) in human ganglion cell layer (n = 4). *: P < 0.05, ****: P < 0.0001; ns, no significant difference.

Low-Dose Trans-RSV Promoted TyrRS Binding to c-Jun

To further observe the relationship between the anti-apoptosis effects of trans-RSV and TyrRS nuclear translocation, the protein that may interact with TyrRS in the nucleus was acquired from the intAct database, and the c-Jun protein of activator protein 1 (AP-1) family of transcription factors was finally screened out. After transfected with FLAG-tagged TyrRS plasmid, the interaction between exogenous TyrRS and c-Jun in HEK293 cells was detected. The trans-RSV promoted exogenous TyrRS binding to c-Jun (Fig. 4A). Endogenous TyrRS were also found to bind to c-Jun following trans-RSV treatment (Figs. 4B, 4C). 

Figure 4.

 

Low-dose trans-RSV promoted TyrRS binding to c-Jun. HEK293 cells were transfected with FLAG-tagged TyrRS plasmid, and the interaction between exogenous (flag-tagged) (A) or endogenous TyrRS (B, C) and c-Jun in HEK293 cells was detected by co-IP assay. All above experiments were repeated three times.

Low-Dose Trans-RSV Inhibited c-Jun Phosphorylation and Inhibited its Transcriptional Activity

Because the transcription factor activity of c-Jun is associated with the phosphorylation of Ser-63 and Ser-73 in the NH2 segment, to observe the c-Jun phosphorylation level in vivo, panretinal proteins of humans and mice were analyzed by Western blot (Figs. 5A, 5B). Diabetes similarly induced decreases in TyrRS levels in both human and mouse retina. The total c-Jun and phosphorylation of Ser-63 were increased in the DM group compared to the control group. Phosphorylation of Ser-73 was not observed in any group. After trans-RSV treatment, increased TyrRS levels were observed, and the total c-Jun level showed no significant difference, but the phosphorylation level of Ser-63 was significantly decreased. The phosphorylation levels of c-Jun were also examined in HEK293 and N2a cells (Figs. 5C, 5D). This result was consistent with the in vivo results. To further explore the downstream phosphorylation of c-Jun Ser-63, we examined the binding affinity of c-Jun and the pro-apoptotic genes BIM, FASL, HRK, and p53 by ChIP‒quantitative PCR (qPCR; Fig. 5E). The binding affinity of c-Jun protein to the corresponding pro-apoptotic genes was decreased, and the total mRNA expression levels of the pro-apoptotic genes were also decreased in HEK 293 cells (Fig. 5F). In summary, low-dose trans-RSV promoted TyrRS binding to c-Jun and inhibited c-Jun phosphorylation and downstream pro-apoptotic gene expression (Fig. 5G). 

Figure 5.

 

Low-dose trans-RSV inhibited c-Jun phosphorylation and inhibited its transcriptional activity. (A) The effects of trans-RSV on TyrRS levels and total and phosphorylated levels of c-Jun were observed by Western blot in the human retina (n = 4). (B) The effects in the mouse retina (n = 8). (C) The effects in N2a cells (n = 6). (D) The effects in HEK293 cells (n = 6). (E) A ChIP assay was performed to detect the capacity of c-Jun to bind to the downstream pro-apoptotic genes BIM, FAS L, HRK, and p53. (F) The qPCR indicated that trans-RSV reduced the mRNA expression levels of pro-apoptotic genes in HEK293 cells. The experiments of E and F were repeated five times. (A *: P 0.01 < P < 0.05, **: P 0.001 < P < 0.01, ***: P < 0.001, ****: P < 0.0001; ns, no significant difference. (G) Schematic diagram showing that low-dose trans-RSV promoted TyrRS binding to c-Jun and affected c-Jun phosphorylation and downstream proapoptotic gene expression.

Discussion

In this study, we confirmed the protective effects of low-dose trans-RSV on diabetes-induced RGC degeneration and HG-induced N2a cell apoptosis. Low-dose trans-RSV promoted TyrRS nuclear translocation both in vitro and in vivo. Importantly, after trans-RSV treatment, endogenous and exogenous TyrRS bound to c-Jun and inhibited the phosphorylation of Ser-63 of c-Jun. Decreased expression of c-Jun-regulated pro-apoptotic genes was observed. These results suggest that low-dose trans-RSV ameliorates diabetes-induced RGC degeneration, and this protective effect might occur via the TyrRS/c-Jun pathway. 

In a previous study, we found that the concentration of RSV that reached the retina after oral administration might be approximately 10 to 100 nmol/L.¹⁸ In this study, we found that this dose might be sufficient to trigger the activation of relevant protective pathways in the retina. It is important to note that increasing the concentration of RSV does not enhance its protective effect but rather causes harm. Previous studies have found that low-dose trans-RSV played a role of caloric restriction in obese patients, delayed cognitive impairment in patients with AD, and postmenopausal women, however, higher than 200 mg/day of trans-RSV leaded to cognitive decline in patients with AD and memory deterioration in patients with schizophrenia.^30–34 According to the results of this study, to achieve retinal protective effects of RSV, oral administration might be sufficient to achieve effective concentrations. The low concentration caused by rapid metabolism promotes its protective effects for its avoidance of the side effects of high concentrations. The interaction of RSV with TyrRS was regarded as an example of xenohormesis through interactions of a natural ligand with a protein target.¹² Xenohormesis is a term to describe the phenomenon in which plants synthesize small molecules or metabolites under mild stress, and these molecules activate a response in heterotrophs, which allows them to survive under stress.³⁵ 

The phenolic rings of RSV and tyrosine have the same disposition in the respective cocrystals; theoretically, RSV could substitute tyrosine for binding to the active site of TyrRS. Sajish et al. demonstrated the binding of RSV and TyrRS in detail.¹² A hexapeptide motif in the anticodon recognition domain of TyrRS was identified as a nuclear localization signal (NLS).¹¹ Mutation in the NLS region can promote or inhibit the nuclear translocation of TyrRS. RSV directly binds to the active site of TyrRS, thereby promoting TyrRS translocation to the nucleus.¹¹ The nuclear function of TyrRS is complicated. Poly (ADP-ribose) polymerase family member 1 (PARP1),¹² a single-stranded DNA damage repair enzyme, and E2F1,¹¹ a transcription factor against DNA damage, were confirmed to be active by endonuclear TyrRS. In this study, we observed the effects of TyrRS nuclear translocation on the phosphorylation of the transcription factor c-Jun and revealed its protective effects directly against apoptosis. 

Strikingly, the nuclear translocation of TyrRS is mainly observed in the GCL. This may be related to the local concentration of RSV or the characteristics of different cells. At present, in addition to genetic diseases, such as Charcot-Marie-Tooth disease, variation of TyrRS in the retina have only been reported in protein profiling of the retinal ischemia and the STZ diabetic rat model.^36,37 Much remains unknown about the non-catalytic function of TyrRS on the retina. This may be a potential research direction in the future. In the ERG, we observed that RSV also had a protective effect on the decrease in a-wave and b-wave amplitudes and the prolongation of implicit time caused by diabetes. This suggests that the protective effects of RSV against diabetic damage may cover the whole retina. The mechanism of these effects in other types of retinal cells remains to be further explored. 

In conclusion, orally taken up trans-RSV can reach RGCs in low doses, promote TyrRS nuclear translocation, inhibit c-Jun phosphorylation, and reduce the expression of pro-apoptotic genes, therefore inhibiting nerve cell apoptosis (Fig. 6). 

Figure 6.

 

Schematic diagram of low-dose trans-resveratrol ameliorates diabetes-induced retinal ganglion cell degeneration via TyrRS/c-Jun pathway. Schematic diagram of trans-RSV reaching RGCs at a low dose, promoting TyrRS nuclear translocation, inhibiting c-Jun phosphorylation, and reducing the expression of pro-apoptotic genes.

Acknowledgments

The authors thank the Center for Biomedical Research, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, for providing technical support. 

Supported by grants from the National Natural Science Foundation of China (81974136) and the Hubei Natural Science Foundation (2020CFB208). 

Disclosure: K. Xiao, None; X.-H. Ma, None; Z. Zhong, None; Y. Zhao, None; X.-H. Chen, None; X.-F. Sun, None