trimetazidine protects retinal ganglion cells from acute ... · trimetazidine protects retinal...
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Trimetazidine protects retinal ganglion cells from acute glaucoma via the
Nrf2/Ho-1 pathway
Peixing Wan, MD*1
; Wenru Su, MD, PhD*1
; Yingying Zhang, MD1; Zhidong Li, MD
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Caibin Deng, BD1; Yehong Zhuo, MD, PhD
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State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen
University, Guangzhou 510060, China.
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These authors contributed equally to this work.
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# Correspondence to:
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Yehong Zhuo, MD,PhD, Phone: +86-020-87331382, Fax: +86-020-87333271, Email:
Wenru Su, MD, PhD, Phone: +86-020-87330402, Fax: +86-020-87333271, Email:
Author contributions: P.W.: conception and design, data collection and assembly, manuscript
writing, and final manuscript approval; W.S.: conception and design, data collection and assembly,
manuscript writing, final manuscript approval, and financial support; Y.Zhang, Z.L. and C.D.: data collection
and assembly; Y.Zhuo: conception and design, manuscript writing, final manuscript approval.
Conflicts of interest: The authors declare that there are no competing financial conflicts of interest.
Financial support: This study is supported by NSFC (81470627). The sponsor of the study had no role in
the design of the original study protocol, in the collection, analysis and interpretation of the data, in
writing the report, or in the decision to submit the manuscript for publication.
Key words: Trimetazidine; Retinal ganglion cells; Acute glaucoma; Oxidation; Inflammation
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10.1042/CS20171182. Please cite using the DOI 10.1042/CS20171182http://dx.doi.org/up-to-date version is available at
encouraged to use the Version of Record that, when published, will replace this version. The most this is an Accepted Manuscript, not the final Version of Record. You are:Clinical Science
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ABSTRACT 26
Acute glaucoma is one of the leading causes of irreversible vision impairment characterized 27
by the rapid elevation of intraocular pressure and consequent retinal ganglion cell (RGC) 28
death. Oxidative stress and neuro-inflammation have been considered critical for the 29
pathogenesis of RGC death in acute glaucoma. Trimetazidine (TMZ), an anti-ischemic drug, 30
possesses anti-oxidative and anti-inflammatory properties, contributing to its therapeutic 31
potential in tissue damage. However, the role of TMZ in acute glaucoma and the underlying 32
molecular mechanisms remain elusive. Here, we report that treatment with TMZ significantly 33
attenuated retinal damage and RGC death in mice with acute glaucoma, with a significant 34
decrease in reactive oxygen species (ROS) and inflammatory cytokine production in the retina. 35
Furthermore, TMZ treatment directly decreased ROS production and rebalanced the 36
intracellular redox state, thus contributing to the survival of RGCs in vitro. TMZ treatment 37
also reduced the production of inflammatory cytokines in vitro. Mechanistically, the 38
TMZ-mediated inhibition of apoptosis and inflammatory cytokine production in RGCs 39
occurred via the inhibition of the nuclear factor erythroid 2-related factor 2/heme oxygenase 40
1/caspase-8 pathway. Moreover, the TMZ-mediated neuroprotection in acute glaucoma was 41
abrogated when an HO-1 inhibitor, SnPP, was used. Our findings identify potential 42
mechanisms of RGC apoptosis and proposea novel therapeutic agent, TMZ, which exerts a 43
precise neuroprotective effect against acute glaucoma. 44
CLINICAL PERSPECTIVE 45
Acute glaucoma jeopardizes normal vision due to substantially high intraocular pressure46
(IOP) and consequent retinal ganglion cell (RGC) death; currently, an attractive47
alternative for next-generation glaucoma therapy is to select an effective drug to prevent48
RGC apoptosis in IOP-induced retinal damage.49
This study demonstrated that trimetazidine (TMZ) significantly ameliorated high50
IOP-induced retinal damage and RGC apoptosis, by exerting therapeutic efficacy through51
its anti-oxidative and anti-inflammatory properties via the Nrf2/HO-1 pathway.52
Our findings suggested that TMZ can protect against acute glaucoma and emerge as a53
promising candidate in the treatment of acute glaucoma and other neurological disorders.54
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INTRODUCTION
Acute glaucoma is one of the main causes of irreversible visual impairment and
blindness, especially among people of Asian descent [1,2]. Acute glaucoma is
characterized by considerable high intraocular pressure (IOP), pain and vision loss [3].
The rapid increase and decrease of IOP causes retinal ischemic/reperfusion (I/R)
injury, thus resulting in apoptosis of retinal ganglion cells (RGCs). Although the
pathogenesis of RGC death is not fully understood, oxidative stress and neuro-
inflammation have been considered critical for the pathogenesis for RGC death in retinal
I/R injury during acute glaucoma [4]. Current treatments that lower IOP through
medication and surgery cannot completely prevent the progressive death of RGCs in acute
glaucoma. Therefore, safe and effective novel alternatives are needed for acute glaucoma
treatment.
Trimetazidine (TMZ), a piperazine-derived antianginal agent [5,6], has traditionally
been used as an anti-ischemic drug for coronary artery disease. Recently, studies have
shown that TMZ possesses anti-oxidative and anti-inflammatory properties and plays
cytoprotective roles in various tissues, including nervous tissue, pancreatic tissue and
renal tissue [7,8]. Therefore, TMZ may be a potential therapeutic alternative for acute
glaucoma treatment. However, the role of TMZ in acute glaucoma has not yet been
explored. Moreover, the mechanisms by which TMZ mediates anti-oxidative and anti-
inflammatory effects remain elusive. Thus, in this study, we investigated the therapeutic
effect of TMZ on experimental acute glaucoma and the anti-oxidative and anti-inflammatory
mechanisms of TMZ.
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MATERIALS AND METHODS 76
Establishment of the retinal I/R model 77
Six- to eight-week-old C57BL/6J male mice were purchased from Guangdong Medical 78
Laboratory Animal Center. The feeding and administration of mice strictly complied with the 79
Association for Research in Vision and Ophthalmology Statement for the Use of Animals in 80
Ophthalmic and Vision Research. 81
The mice were anesthetized with 100 mg/kg pentobarbital sodium by intraperitoneal injection. 82
Before operation, mice corneas were topically anesthetized with 0.5% Alcaine eye drops, and 83
pupils were dilated with 1% Tropicamide. We then conducted cannulation into the anterior 84
chamber of the right eye using a 30-gauge needle supplied with balanced salt solution to 85
maintain the IOP at 70 mmHg for 60 minutes. Sham operation was performed in the 86
contralateral eye without elevating the IOP to serve as the control. After 60 minutes, the IOP 87
was normalized by withdrawing the needle, and then tobramycin ointment was used to 88
prevent bacterial infection. 89
Preparation of Trimetazidine 90
TMZ with purity higher than 95% was purchased from Sigma Chemical Co. (St Louis, USA), 91
dissolved in sterile PBS to a stock concentration of 0.1 mol/L, and stored at 4°C in the dark to 92
be used within 2 days after preparation. 93
Experiment Grouping Design 94
The mice were randomly divided into five groups: normal control group (Normal), Tin 95
protoporphyrin IX dichloride (SnPP) group, I/R group, TMZ (100 μM) group and TMZ + 96
SnPP group. The mice in I/R, TMZ and TMZ + SnPP groups were subjected to experimental 97
I/R. The normal control and SnPP groups had sham cannulation performed without elevating 98
the IOP as described above. The eyes of mice in the SnPP or TMZ groups were intravitreally 99
injected with 1 μL TMZ [9] or SnPP solution, respectively, before the onset of reperfusion. 100
The eyes of mice in the TMZ + SnPP group were intravitreally injected with 1 μL TMZ and 101
SnPP solution together before the onset of reperfusion. The eyes of mice in the normal control 102
group were injected with 1 μL of sterile PBS into the vitreous cavity. 103
RGC Labeling and Survival Quantification. 104
Mice were anesthetized with 100 mg/kg pentobarbital sodium by intraperitoneal injection and 105
placed in a stereotactic apparatus (Stoelting, USA). The skull was exposed and cleaned with 106
PVP-J. Bilateral holes were drilled at the surface of superior colliculi. Approximately 1 µl of 107
4% Fluorogold (FG, hydroxystilbamidine; Fluorochrome, USA) solution was injected into 108
both superior colliculis. After injection, the micro-syringe was kept still for 30 seconds and 109
then slowly removed. Finally, the dissected scalp was sutured with topical application of 110
tobramycin. 111
To ensure proper RGC labeling, the animals were allowed 7 days for retrograde transport of 112
FG before sacrifice. FG-positive RGCs were identified with a fluorescence microscope 113
(AxioImager; Carl Zeiss MicroImaging Inc., USA) in retinal flat mount. Surviving RGCs 114
(green dots) were counted automatically using ImageJ (LOCI, University of 115
Wisconsin-Madison). 116
Histological examination 117
Seven days after I/R treatment, mouse eyes were enucleated and embedded in paraffin. Every 118
paraffin block was sectioned to 4-µm thickness through the optic nerve. Three sections of 119
each eye were cut and stained with hematoxylin and eosin (H&E). The inner plexiform layer 120
(IPL) thickness was measured within 1 mm to the optic nerve center to quantify retinal 121
damage by Axiovision software (Carl Zeiss MicroImaging Inc.). The data from three sections 122
per eye were averaged. 123
Primary culture of RGCs 124
Primary RGCs were obtained according to the protocol published by Alissa Winzeler and 125
Jack T. Wang in 2013 [10]. In brief, retinal samples were separated from neonatal mice to 126
prepare single cell suspensions. The retinal suspension was incubated in rabbit anti-mouse 127
macrophage antibody-coated flasks (Cedarlane, USA) and goat anti-mouse macrophage 128
antibody-coated flasks (Jackson Immuno Research, USA) to remove the adherent 129
macrophages. The non-adherent cells were transferred to Thy1.2 monoclonal antibody-coated 130
flasks (Millipore Chemicon, USA) to collect adherent cells. The adherent RGCs were 131
incubated at 37°C in 5% CO2 with RGC Growth Medium containing supplements as 132
described. 133
Mix culture with BV2 cells 134
Mouse microglia cell line BV2 (ATCC, USA) was co-cultured with primary RGCs in DMEM 135
(High Glucose 4.5 g/ml, Gibco, USA) supplemented with 10% fetal bovine serum (FBS) at 136
37°C with 5% CO2 and 95% air. RGCs were placed on the upper permeable membrane of a 137
transwell chamber, with BV2 cells grown in the lower well of the plate for 24 hours before 138
treatment. 139
Establishment of the Oxygen-glucose deprivation and reperfusion (OGD/R) model 140
The OGD/R model was established as follows: the culture medium was replaced with 141
glucose-free DMEM (Gibco) after washing the cells twice with PBS, and then the cells were 142
placed in a modular incubator chamber (Billups-Rothenberg, Inc., Del Mar, CA) filled with a 143
gas mixture of 5% CO2 and 95% N2 at 37°C for 3 hours. The control cells were incubated in 144
serum-free medium with 4.5 g/L D-glucose under normoxic conditions (5% CO2 and 95% air) 145
for the same duration. At the end of the exposure period, the cells were returned to normoxic 146
conditions with glucose and incubated for 12 hours. 147
Cell treatment with TMZ 148
Primary RGCs were cultured in 6-well plates or transwell plates before treatment. TMZ was 149
added at indicated concentrations into cell supernatants 6 hours prior to the onset of OGD/R 150
or other assays. TMZ was diluted in DMEM and added at concentrations ranging from 0.1 to 151
100 μM. 152
Enzyme-linked Immunosorbent Assay (ELISA) 153
The cytokines TNFα and IL1β were measured in the culture supernatant of BV2 cells. 154
Immunochemical analyses were conducted using commercially available ELISA kits 155
(eBioscience, Vienna, Austria). For measuring the level of 3-nitrotyrosine proteins in retinal 156
homogenates, a commercial kit was used (Oxiselect Nitrotyrosine kit; Cell Biolabs, Inc., 157
USA), following the manufacturer’s instructions. 158
Viability and Apoptosis Assays in RGCs 159
Cell viability was measured using the CCK8 Assay Kit (Beyotime Biotechnology, China) 160
according to the manufacturer’s protocol. CCK8-reduction activity was presented as the 161
percentage of the unexposed control cells (100%). Flow cytometry was also performed to 162
measure OGD/R-induced apoptosis of RGCs by using a propidium iodide (PI) and Annexin 163
V-FITC detection kit (BD Biosciences, New York, USA) according to the manufacturer’s 164
instructions. Flow cytometric analysis using FlowJo 7.6.2 (FlowJo, LLC) was performed 165
following standard protocols. 166
Inhibition of Ho-1 Activity 167
Ho-1 activity was inhibited in vivo through an intraperitoneal injection of SnPP (40 mol/kg, 168
Tocris Bioscience, USA) 0.5 h prior to I/R and once daily for 3 days after I/R. SnPP was 169
dissolved in 0.1 N NaOH and diluted with PBS (pH = 7.4). 170
Cells were seeded in a 6-well plate at approximately 70% confluency. Ten micromolar SnPP 171
or vehicle was added to the culture medium for 6 hours. The cells were then treated with TMZ 172
or vehicle for 6 hours and subjected to OGD/R. 173
Detection of Intracellular ROS Levels 174
Quantification of intracellular reactive oxygen species (ROS) accumulation was performed by 175
fluorescence detection as well as flow cytometry using the fluorescent probe 176
2′,7′-dichlorofluorescein diacetate (DCFH-DA, KeyGEN, China). Primary RGCs were 177
subjected to the appropriate treatments and then incubated for 20 minutes in the dark at 37°C 178
with 10 µM DCFH-DA solutions. After incubation, the cells were analyzed within 30 minutes. 179
Mean fluorescence intensity of ROS was measured using a fluorescence microscope, and flow 180
cytometry was performed using a Fortessa system (BD). The data were analyzed using 181
ImageJ and FlowJo software. 182
Analysis on MMP (JC-1 staining) 183
The changes in mitochondrial membrane potential (MMP) in RGCs were explored using the 184
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolyl carbocyanine iodide (JC-1) probe as 185
described by the manufacturer and then visualized using an inverted fluorescence microscope. 186
The average result of three plates of cells or mice were obtained for each condition. 187
Quantitative Real-time PCR 188
Total RNA was extracted from the retinal samples and cultured cells by TRIzol reagent 189
(Invitrogen, USA) following the manufacturer’s protocol. cDNA synthesis was conducted 190
with PrimeScript RT Master Mix (TaKaRa, China). Quantitative analysis was performed with 191
a Light Cycler 480 Real-Time PCR System. The expression level of target mRNA was 192
measured and normalized to Gapdh. The primer sequences are as follows: GAPDH-forward 193
primer CCGGGAAACTGTGGCGTGATGG; GAPDH-reverse primer 194
AGGTGGAGGAGTGGGTGTCGCTGTT; TNFα-forward primer 195
GCACCACCATCAAGGACTCAA; TNFα-reverse primer TCGAGGCCCAGTGAATTCG; 196
IL1β -forward primer GGGCCTCAAAGGAAAGAATC; IL1β-reverse primer 197
CTCTGCTTGTGAGGTGCTGA. 198
Western Blot 199
Total protein was isolated from the retinal samples and cultured cells according to standard 200
procedures. Nuclear protein was extracted using ProteoExtract® Subcellular Proteome 201
Extraction Kit (Merck Millipore, German) following the manufacturer’s protocol. Proteins 202
were run on 12% polyacrylamide gels following standard protocol. The expression of total 203
protein and nuclear protein was normalized to Gapdh and Histone H3, respectively, and then 204
quantified using ImageJ. 205
The primary antibodies and dilutions used were as follows: anti-Nrf2 (CST, USA, 1:400), 206
anti-Ho-1 (CST, 1:200), anti-cleaved-Caspase-8 (CST, 1:400), anti-Gapdh (CST, 1:400), 207
anti-Histone H3 (Abcam, UK, 1:400). 208
Caspase-8 Activity Assay 209
The Caspase-8 activity of retinal tissues and RGCs were performed using the CaspGLOWTM
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Fluorescein Active Caspase-8 Staining Kit (BioVision, Milpitas, USA) according to the 211
manufacturer’s instructions. One microliter of FITC-IETD-FMK was added into each tube 212
containing 300 µl of cell suspension and incubated for 1 hour at 37°C in 5% CO2. Then, the 213
cells were resuspended, and the fluorescence intensity was measured at Ex/Em = 485/535 nm. 214
Statistical Analysis 215
The data were presented as the mean ± standard deviation (SD). One-way analysis of variance 216
(ANOVA) was performed, followed by Bonferroni’s post hoc test using SPSS software, 217
version 17 (SPSS Inc., Chicago, IL, USA). All statistical tests were two-tailed, and a p-value 218
below 0.05 was considered statistically significant. 219
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RESULTS 221
Neuroprotective effects of intravitreal TMZ in IOP-induced retinal damage 222
To evaluate the role of TMZ in acute glaucoma, TMZ was administered intravitreally before 223
acute elevation of IOP. We first investigated the change in retinal thickness in three 224
independent groups of mice. Retinas exposed to I/R displayed a significant decrease in IPL 225
thickness compared with normal retinas 7 days after reperfusion. However, the damage to the 226
retina, especially to the IPL, was significantly ameliorated by TMZ (Fig. 1A and 1B). The 227
irreversible apoptosis of RGCs is another important indicator for the functional damage of the 228
retina. FG analysis demonstrated that the number of RGCs was decreased in the experimental 229
mice at 7 days after reperfusion compared with that observed in control mice (Fig. 1C and 230
1D). Intravitreal TMZ injections significantly decreased the severity of retinal damage and 231
extent of RGC death (Fig. 1C and 1D). Previous studies have shown that over-production and 232
accumulation of neurotoxic mediators, including ROS, TNF-α and IL-1β, play important roles 233
in the primary and secondary waves of RGC apoptosis [11-13]. As shown in Fig. 1E, the 234
production of nitrotyrosine (a protein derivative of ROS) was significantly increased in the 235
whole retina at 24 hours after reperfusion compared with that in control retinas, and it 236
declined in the retinas treated with TMZ. In addition, intravitreal injection of TMZ 237
significantly suppressed the expression of TNFα and IL1β mRNA (Fig. 1F and 1G). 238
Collectively, these data show that TMZ exerts neuroprotective effects against IOP-induced 239
retinal damage. 240
TMZ directly protects RGC apoptosis induced by OGD/R 241
Next, we performed a series of in vitro studies to explore whether TMZ is capable of directly 242
inhibiting the apoptosis of RGCs and the underlying mechanisms. We first identified primary 243
cultured RGCs by characteristic markers. Immunostaining results indicated that primary 244
RGCs indeed expressed Brn-3a, β3-tubulin and RBPMS (Fig. 2A). CCK8 assay was used to 245
test whether TMZ can influence cellular metabolism or proliferation. The results showed that 246
cellular metabolism was affected when TMZ concentration reached 50 μM (Fig. 2B). 247
Therefore, we selected the concentration range from 0.1 to 10 μM for subsequent in vitro 248
studies. We then exposed primary RGCs to OGD/R to effectively mimic the IOP-induced I/R 249
damage in vivo [4,14]. OGD/R decreased cell viability, and TMZ (0.1-10 μM) treatment 250
significantly increased the number of surviving RGCs (Fig. 2C). The flow cytometry results 251
showed that the number of apoptotic cells marked by PI and AnnexinV was increased after 252
OGD/R exposure, whereas TMZ treatment significantly decreased the number of 253
PI+Annexin+ cells (Fig. 2D and 2E). The TMZ concentration range from 1 to 10 μM was 254
found to be the most effective in exerting cytoprotective effects against OGD/R. In addition, 255
TMZ significantly decreased caspase-8 activation as measured by Western blotting and 256
caspase-8 activity assay (Fig. 2F and 2G). These results demonstrate that TMZ decreases 257
OGD/R-induced RGC apoptosis in vitro. 258
The anti-oxidant effect of TMZ is involved in the TMZ-mediated neuroprotection in 259
RGCs 260
Primary RGCs in the OGD/R group display a greater extent of ROS production than the cells 261
in the control group. TMZ was found to significantly attenuate the over-production of ROS in 262
RGCs exposed to OGD/R as marked by the reduction in green fluorescence in a 263
concentration-dependent manner (Fig. 3A and 3B). The diminished ROS production in 264
primary RGCs by TMZ could also be observed by flow cytometry. The peak area occupied by 265
FITC+ cells, representing cells with excess ROS, was found to be significantly decreased by 266
TMZ treatment (Fig. 3C and 3D). OGD/R also resulted in a decrease in the MMP in RGCs, 267
which was marked by fluorescence shift from red to green. However, TMZ treatment 268
protected mitochondrial function by restoring the MMP (Fig. 3F and 3G). These results 269
further support the anti-oxidant effect of TMZ on RGCs in vitro. 270
TMZ conferred protection against apoptosis in RGCs via Nrf2/Ho-1 signaling 271
The above results showed that the anti-oxidant effects of TMZ are actively involved in the 272
TMZ-mediated protection of RGCs. Nrf2, a transcription factor, is regarded as a master 273
regulator of cellular responses to oxidative stress. Thus, we next determined whether Nrf2 and 274
its downstream signaling factor, Ho-1, is associated with the TMZ-mediated protection 275
against RGC apoptosis. The Western blotting results showed that TMZ (10 μM) treatment 276
significantly induced the nuclear translocation of Nrf2 in RGCs under OGD/R conditions (Fig. 277
4A). TMZ treatment also increased Ho-1 protein expression compared with vehicle treatment 278
(Fig. 4B). To further confirm the role of Nrf2/Ho-1 signaling in the TMZ-mediated 279
neuroprotection of RGCs, SnPP, a Ho-1 inhibitor was used. Our results showed that SnPP 280
treatment significantly but incompletely reversed the TMZ-mediated neuroprotection of 281
RGCs (Fig. 4C and 4D). The reduction of intracellular ROS (Fig. 4E and 4F) and alterations 282
in MMP (Fig. 4G and 4H) induced by TMZ were partially inhibited by SnPP. Taken together, 283
these data suggest that TMZ confers neuroprotection through an Nrf2/Ho-1-dependent 284
mechanism. 285
Nrf2/Ho-1 signaling is essential for TMZ-mediated anti-inflammatory effects in vitro 286
We also performed a series of in vitro experiments to further evaluate the anti-inflammatory 287
effects of TMZ and to identify the underlying mechanisms. For this purpose, BV2 cells, an 288
established mouse microglial cell line was cultured under OGD/R conditions. BV2 cells were 289
plated in medium in the presence or absence of TMZ for 6 h before OGD/R. We used ELISA 290
kits to examine the concentration of TNFα and IL1β in the supernatant of BV2 cells after 291
OGD/R. The OGD/R-induced over-production of TNFα and IL1β could be inhibited by TMZ 292
(Fig. 5A). The real-time PCR results further confirmed the anti-inflammatory effects of TMZ 293
(Fig. 5B). Nrf2/Ho-1 signaling plays an important role in the TMZ-mediated neuroprotection 294
of RGCs. Thus, we next assessed whether this pathway is also involved in the 295
anti-inflammatory effects of TMZ. As shown in Fig. 5C and 5D, TMZ significantly induced 296
the nuclear translocation of Nrf2 and increased the protein expression of Ho-1 in BV2 cells 297
under OGD/R conditions. Our results demonstrated that SnPP significantly but incompletely 298
reversed the TMZ-mediated inhibition of TNFα and IL1β production (Fig. 5E and 5F). In 299
addition, the inhibitory effect of TMZ on caspase-8 activation was significantly decreased 300
after SnPP treatment (Fig. 5G). These results indicate that TMZ is capable of regulating 301
Nrf2/Ho-1 signaling to exert anti-inflammatory effects in vitro. 302
TMZ protects RGCs from IOP-induced damage via Nrf2/Ho-1 signaling 303
Our in vitro studies showed that Nrf2/Ho-1 signaling plays a critical role in TMZ-mediated 304
anti-oxidant and anti-inflammatory effects. Therefore, we determined whether Nrf2/Ho-1 305
signaling was also involved in the TMZ-mediated neuroprotection against IOP-induced retinal 306
damage. The Western blotting results showed that TMZ treatment significantly induced the 307
nuclear translocation of Nrf2 and increased the expression of Ho-1 in the retinas (Fig. 6A and 308
B). SnPP was intraperitoneally injected (40 mol/kg) 0.5 h prior to I/R and once daily for 3 309
days after I/R. As shown in Fig. 6C-F, treatment with the Ho-1 inhibitor significantly blocked 310
the TMZ-mediated neuroprotection against IOP-induced retinal damage. The Ho-1 inhibitor 311
also significantly decreased the inhibitory effect of TMZ on ROS accumulation and 312
inflammatory cytokine production (Fig 6G-I). These findings suggest that the upregulation of 313
Nrf2/Ho-1 signaling may contribute, at least in part, to the TMZ-mediated neuroprotection 314
against IOP-induced retinal damage. 315
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DISCUSSION
Acute glaucoma jeopardizes normal vision due to substantially high IOP. The progressive loss
of RGCs caused by rapid increase of IOP is mainly responsible for irreversible blindness in
acute glaucoma [15]. Currently, selecting effective drugs to prevent RGC apoptosis in
IOP-induced retinal damage has become an attractive alternative for next-generation
glaucoma therapy. TMZ, which improves energy metabolism in ischemic myocytes, was first
developed as an anti-angina drug [16]. However, a growing number of studies suggest that
TMZ possesses numerous non-antianginal functions, including immunosuppression [17],
anti-oxidation [18], anti-ischemia [19,20] and anti-apoptosis [21]. This study
demonstrated that treatment with TMZ significantly ameliorated high IOP-induced retinal
damage and RGC apoptosis, with a significant decrease in ROS and inflammatory cytokine
production in the retina. TMZ primarily exerts its therapeutic efficacy through its
anti-oxidative and anti-inflammatory effects. These results are consistent with those of
previous studies, showing that TMZ treatment attenuates acute inflammatory responses and
oxidative stress in experimental cardiac remodeling [22] and pancreatitis [23]. Next, we
found that the anti-oxidative and anti-inflammatory effects of TMZ depended primarily on the
Nrf2/HO-1 pathway. Interestingly, TMZ treatment in IOP-induced retinal damage resulted
in an obvious activation of the Nrf2/HO-1 pathway, which plays a crucial role in TMZ-
mediated protection against IOP-induced retinal damage. These findings suggest that
TMZ exerts neuroprotective effects against acute glaucoma mainly through anti-
oxidative and anti-inflammatory properties.
The transcription factor Nrf2 is a central regulator of cellular responses to oxidative stress
stimulation [24]. Under normal conditions, Nrf2 is an inactive complex in the cytoplasm with
Kelch-like ECH-associated protein 1. When cells undergo oxidative stress, the complex is
disrupted, and, subsequently, Nrf2 translocates to the nucleus, leading to target gene
transcription, including Ho-1, a key heme-degrading enzyme [25,26]. In addition to its
well-known anti-oxidative properties, the Nrf2/Ho-1 signaling pathway also exhibits
anti-inflammatory and anti-apoptotic proprieties [27,28]. In this study, we demonstrated that
TMZ treatment promoted the nuclear translocation of Nrf2 and subsequently increased the 345
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expression of HO-1 in primary RGCs and BV2 cells. Furthermore, HO-1 blockade
significantly but incompletely reversed the TMZ-mediated anti-oxidative and
anti-inflammatory effects. Importantly, the HO-1 inhibitor also blocked the TMZ-mediated
neuroprotection in RGCs in acute glaucoma. Therefore, these compelling findings suggest
that Nrf2/HO-1 signaling contributes, at least in part, to the TMZ-mediated neuroprotection in
acute glaucoma.
Caspase-8, a well-characterized initiator of apoptotic signaling, has been shown to have
multiple non-apoptotic functions. Burguillos et al. reported that caspase-8 promotes
microglial activation and neuroinflammation. Our recent study indicated that caspase-8
mediates the production of cytokines, including IL1β and TNFα in immunocytes, which play
important roles in high-IOP-induced retinal damage [4]. In this study, we observed that TMZ
inhibited caspase-8-mediated RGC apoptosis. Furthermore, TMZ reduced caspase-8-
dependent inflammatory reactions both in vivo and in vitro. Therefore, combining our results
on the protective role of TMZ in acute glaucoma with existing evidence enables us to propose
a mechanistic model that is described in Fig. 7 Proposed model for the neuroprotective
mechanisms of TMZ in acute glaucoma.
The unique pharmacological properties of TMZ enhance its efficacy and enable it to reach its
peak concentration in less than 2 h. Timely treatment is closely associated with efficacy of
neuroprotection [1,2]. Hence, TMZ could be used to halt the early stages of RGCs apoptosis
in acute glaucoma, which is attributable to its fast mechanism of action. Due to its efficacy
and safety, TMZ could be used a promising candidate to treat acute glaucoma and other
related ocular diseases.
In summary, our study initially demonstrated the neuroprotective effects of TMZ on
experimental acute glaucoma. Our results showed that TMZ inhibited RGC apoptosis and
inflammation in vitro and in vivo, possibly by inhibiting caspase-8 signaling. Importantly, we
further determined the neuroprotective mechanisms of TMZ by demonstrating that
Nrf2-HO-1 signaling is essential for TMZ-mediated neuroprotective effects in vitro and in
vivo. These findings provide compelling evidence that TMZ can protect against acute
glaucoma and emerge as a promising candidate in the treatment of acute glaucoma. 374
References375
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445
446
Figure legends 447
448
449
RG
C s
ur
viv
al
(%
)
No
r ma
l
I / R
TM
Z
0
5 0
1 0 0 * *
C D
Normal I/R TMZ
Me
an
th
ick
ne
ss
of
IPL
(μM
)
Norm
a lI /
R
TM
Z
1 0
2 0
3 0
4 0
**
GCLIPL
INL
ONL
A B
Figure 1
Nit
roty
rosin
e
(
M)
Norm
a lI /
R
TM
Z
0
3 0
6 0
9 0
**
TN
F
mR
NA
(F
old
ch
an
ge
)
No
r ma
l
I / R
TM
Z
0
2
4
6* *
IL
1
mR
NA
(F
old
ch
an
ge
)
No
r ma
l
I / R
TM
Z
0
2
4
6
* *
E F G
Figure 1
Normal I/R TMZ
Figure 1. Neuroprotective effects of intravitreal TMZ against IOP-induced retinal 450
damage 451
A and B. H&E staining of retinal cross-sections showing that intravitreal TMZ injection 452
significantly inhibited the attenuation of total retinal thickness and IPL thickness in response 453
to I/R damage 7 days after reperfusion. 454
C and D. FG labeling was performed 7 days after reperfusion. The results indicated that TMZ 455
significantly increased the number of surviving cells in the RGC layer compared with that in 456
the untreated I/R group. 457
E. The production of nitrotyrosine was significantly decreased by TMZ treatment in the whole 458
retina 24 hours after reperfusion compared with that in the untreated retinas. 459
F and G. As measured by real-time PCR, intravitreal injection of TMZ significantly 460
suppressed the expression of TNFα and IL1β mRNA in retinas. 461
GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer 462
nuclear layer. Scale bar = 50 μm. 463
The data represents the means ± SD (n=6). *p < 0.05,
**p < 0.01. 464
465
466
467
Annexin
PI
0 0.1 1 10 μM
T M Z ( M )
Ce
ll
Su
rv
iva
l
(%
)
0
0. 1 1
10
50
10
0
5 0
7 5
1 0 0
*
RBMPS β3-tublin
Brn
3a
/DA
PI
Ce
ll s
urv
iva
l
(%)
0 00.1 1 1
0
2 5
5 0
7 5
1 0 0
O G D /R + T M Z (M )
**
**
A B C
D E
Figure 2
Re
la
tiv
e d
en
sity
00
. 1 11
00 . 0
0 . 2
0 . 4
0 . 6* *
* *
T M Z ( M )
CleavedCasp8
Gapdh
Ca
sp
8 a
ctiv
ity
(F
old
ch
an
ge
)
00
. 1 11
00 . 0 0
0 . 2 5
0 . 5 0
0 . 7 5
1 . 0 0
* *
* *
T M Z ( M )
F G
PI
+A
nn
ex
in+
Ce
lls
(%
)
00
. 1 11
00
5
1 0
1 5
2 0 * *
*
T M Z ( M )
15.8% 4.17% 3.15% 2.93%
43kDa
37kDa
Figure 2. TMZ directly protects RGCs against apoptosis induced by OGD/R 468
A. Immunostaining results indicated that primary RGCs indeed expressed characteristic 469
markers of retinal ganglion cells including Brn-3a (red), RBPMS (green, left) and β3-tubulin 470
(green, right). 471
B. CCK8 assay showed that cellular metabolism was affected when TMZ concentration 472
reached 50 µM. 473
C. CCK8 assay showed that cell survival was significantly increased by TMZ treatment after 474
OGD/R. 475
D and E. The results of flow cytometry showed that the proportion of apoptotic cells was 476
increased to 15.8% (± 3.1%) after OGD/R exposure. TMZ significantly decreased the 477
proportion of PI+Annexin+ cells to an average of 2.93%. 478
F. Western blot analysis demonstrated that TMZ significantly suppressed the level of 479
cleaved-caspase-8. 480
G. The reduction of caspase-8 activity was confirmed by caspase-8 activity assay. 481
Scale bar = 20 μm. The data represent the means ± SD (n=8). *p < 0.05, **p < 0.01. 482
483
484
485
ROS
Cel
l C
ou
nt
TMZ 0 0.1 1 10 μM
86.1% 57.1% 45.4% 28.3%
C D
RO
S+
Ce
lls
(%
)
00
. 1 11
00
3 0
6 0
9 0* *
* *
T M Z ( M )
Figure 3
TMZ 0 0.1 1 10 μM
TMZ 0 0.1 1 10 μM
RO
S+
Ce
lls
(N
um
be
r/m
m2
)
00
. 1 11
00
2 0 0
4 0 0
6 0 0 * *
* *
T M Z ( M )
PE
/F
IT
C
00
. 1 11
00
1
2
3
4
* *
* *
T M Z ( M )
A B
E F
Figure 3. The anti-oxidant effect of TMZ is involved in the TMZ-mediated 486
neuroprotective effect on RGCs 487
A and B. After OGD/R exposure, primary RGCs were subjected to ROS over-production 488
(green fluorescence). TMZ could significantly decreasing ROS levels in RGCs. 489
C and D. As marked by the peak area of FITC+ population, the proportion of cells with 490
excess ROS was significantly decreased from 80% (± 6.1%) to 30% (± 1.7%) in the OGD/R 491
+TMZ RGCs. 492
E and F. OGD/R decreased the MMP, which was marked by a fluorescence shift from red to 493
green in RGCs. TMZ pretreatment elevated the PE/FITC ratio. 494
Scale bar = 50 μm. The data represent the means ± SD (n=8). *p < 0.05, **p < 0.01. 495
496
497
16.92% 18.12 % 3.52% 14.36%
Annexin
PI
OGD/R SnPP TMZ TMZ+SnPP
PI+
An
ne
xin
+(%
)
OG
D/R
SnP
P
TM
Z
0
1 0
2 0
3 0
**
**
T M Z+
S n P P
Figure 4
76.1% 80.34% 22.52 % 58.36 %
ROS
Cel
l co
un
t
OGD/R SnPP TMZ TMZ+SnPP
PE
/FIT
C
OG
D/R
SnP
P
TM
Z
0
1
2
3
****
T M Z+
S n P P
OGD/R SnPP TMZ TMZ+SnPP
RO
S+
Ce
lls (
%)
OG
D/R
SnP
P
TM
Z
0
2 5
5 0
7 5
1 0 0
**
**
T M Z+
S n P P
A B C
D E F
G H
Nrf2
Histone H3
Re
la
tiv
e d
en
sity
OG
D/ R
TM
Z
0 . 0
0 . 3
0 . 6
0 . 9
* *
GapdhR
ela
tiv
e d
en
sity
OG
D/ R
TM
Z
0 . 0
0 . 3
0 . 6
0 . 9* *
Ho-197kDa
17kDa
28kDa
37kDa
Figure 4. TMZ protects RGCs from apoptosis via Nrf2/Ho-1 signaling 498
A and B. Western blot analysis demonstrated that TMZ elevated the nuclear translocation of 499
Nrf2 and total expression of Ho-1 in primary RGCs under OGD/R conditions. 500
C and D. TMZ suppressed OGD/R-induced apoptosis in primary RGCs. SnPP treatment 501
significantly but incompletely reversed TMZ-mediated cytoprotective effect on RGCs. 502
E and F. Flow cytometry assay of ROS accumulation (FITC+) in RGCs. SnPP resulted in an 503
increase in the proportion of FITC+ cells and inhibited the TMZ-induced anti-oxidative 504
effect. 505
G and H. The decreasing of PE/FITC ratio indicated significant disruption of the MMP in the 506
SnPP-treated RGCs, which could not be rescued by TMZ treatment. 507
Scale bar = 50 μm. The data represent the means ± SD (n=8). *p < 0.05, **p < 0.01. 508
509
510
511
nNrf2Histone
H3 Gapdh
Cleaved- casp8
(a) (b) (c)
pg
/ml
OG
D/ R
TM
Z
OG
D/ R
TM
Z
0
5 0
1 0 0
1 5 0 * *
* *
T N F
I L 1
mR
NA
(F
old
ch
an
ge
)
OG
D/ R
TM
Z
OG
D/ R
TM
Z
0 . 0 0
0 . 2 5
0 . 5 0
0 . 7 5
1 . 0 0* * * *
T N F I L 1
Re
la
tiv
e d
en
sity
OG
D/ R
TM
Z
0 . 0
0 . 3
0 . 6
0 . 9
* *
Nrf2
Histone H3 Gapdh
Re
la
tiv
e d
en
sity
OG
D/ R
TM
Z
0 . 0
0 . 3
0 . 6
0 . 9
* *
pg
/ml
OG
D/R
SnP
P
TM
Z
TM
Z+
SnP
P
OG
D/R
SnP
P
TM
Z
TM
Z+
SnP
P0
5 0
1 0 0
1 5 0
**
**
T N F
IL 1
mR
NA
(Fo
ld c
ha
ng
e)
OG
D/R
SnP
P
TM
Z
TM
Z+
SnP
P
OG
D/R
SnP
P
TM
Z
TM
Z+
SnP
P0 .0
0 .5
1 .0
1 .5
****
T N F IL 1
A B C D
E F G
Figure 5
Ca
sp
8 a
cti
vit
y
(Fo
ld c
ha
ng
e)
OG
D/R
SnP
P
TM
Z
TM
Z+
SnP
P
0 .0
0 .5
1 .0
1 .5
**
Ho-197kDa
17kDa
28kDa
37kDa
Figure 5. Nrf2/Ho-1 signaling is essential for the TMZ-mediated anti-inflammatory 512
effects in vitro 513
A. As measured by ELISA, the concentration of TNFα and IL1β in supernatants was found to 514
be significantly reduced by TMZ. 515
B. Real-time PCR analysis showed that TMZ decreased TNFα and IL1β mRNA in the TMZ 516
group. 517
C and D. TMZ significantly induced the nuclear translocation of Nrf2 and increased the 518
protein expression of Ho-1 in BV2 cells under OGD/R conditions 519
E and F. SnPP significantly but incompletely reversed the TMZ-mediated inhibition on TNFα 520
and IL1β production as measured by ELISA and real-time PCR. 521
G. SnPP treatment significantly decreased the inhibitory effect of TMZ on caspase-8 522
activation. 523
The data represent the means ± SD (n=8). *p < 0.05, **p < 0.01. 524
525
526
Nrf2
Histone H3
Re
la
tiv
e d
en
sity
I / R
TM
Z
0 . 0
0 . 3
0 . 6
0 . 9
* *
GapdhR
ela
tiv
e d
en
sity
I / R
TM
Z
0 . 0
0 . 3
0 . 6
0 . 9* *
Me
an
th
ick
ne
ss
of
IPL
( μM
)
I /R
SnP
P
TM
Z
0
1 0
2 0
3 0**
T M Z+
S n P P
RG
C s
urv
iva
l
(%)
I /R
SnP
P
TM
Z
0
5 0
1 0 0 **
T M Z+
S n P P
I/R SnPP TMZ TMZ+SnPP
Figure 6
Nit
roty
rosin
e
(
M)
I /R
SnP
P
TM
Z
0
4 0
8 0
1 2 0 **
T M Z+
S n P P
TN
F
m
RN
A
(Fo
ld c
ha
ng
e)
I /R
SnP
P
TM
Z
0 .0
0 .5
1 .0
1 .5
**
T M Z+
S n P P
IL1
m
RN
A
(Fo
ld c
ha
ng
e)
I /R
SnP
P
TM
Z
0 .0
0 .5
1 .0
1 .5
**
T M Z+
S n P P
A B C
D E F
G H I
I/R SnPP TMZ TMZ+SnPPHo-197kDa
17kDa
28kDa
37kDa
Figure 6. TMZ protects RGCs from IOP-induced damage via Nrf2/Ho-1 signaling 527
A and B. Western blot analysis demonstrated that TMZ dramatically induced the nuclear 528
translocation of Nrf2 and increased the expression of Ho-1 in the retinas. 529
C and D. Treatment with the SnPP significantly blocked the TMZ-mediated neuroprotection 530
in IOP-induced retinal attenuation, especially IPL thickness. 531
E and F. SnPP dramatically inhibited the TMZ-mediated neuroprotection in IOP-induced 532
RGC apoptosis. 533
G. SnPP also significantly decreased the inhibitory effect of TMZ on nitrotyrosine production. 534
H and I. Real-time PCR results confirmed that SnPP significantly suppressed the 535
TMZ-induced decrease in TNFα and IL1β mRNA levels compared with the TMZ only group. 536
537
538
539
Figure 7. Proposed Model for the Neuroprotective Mechanisms of TMZ in Acute 540
Glaucoma 541
I/R damages the function of mitochondria by disrupting the MMP, which leads to the 542
over-production of ROS. ROS pushes cells toward the apoptotic fate and stimulates 543
neuro-inflammation. Nrf2/Ho-1 pathway can be activated by TMZ to confer protective effects 544
by inhibiting the accumulation of ROS. Immunocyte activation during injury results in 545
caspase-8-dependent inflammation by increasing TNFα and IL1β production, which also 546
induces RGC apoptosis. Pro-inflammatory reactions can be effectively suppressed by TMZ. 547
Therefore, RGC apoptosis can be attenuated by pretreatment with TMZ via anti-oxidative and 548
anti-inflammatory mechanisms in acute glaucoma. 549
I/R, ischemia/reperfusion injury; RGC, retinal ganglion cell; MMP, mitochondrial membrane 550
potential; ROS, reactive oxygen species. 551