Artemisinin Protects Retinal Neuronal Cells against Oxidative Stress

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Artemisinin protectes retinal neuronal cells against oxidative stress and restores rat retinal physiological function from light exposed-damage Fengxia Yan, Hai-Tao Wang, Yang Gao, Jiang-Ping Xu, and Wenhua Zheng ACS Chem. Neurosci., Just Accepted Manuscript • Publication Date (Web): 27 Apr 2017 Downloaded from http://pubs.acs.org on April 28, 2017

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ACS Chemical Neuroscience

1

Artemisinin protects retinal neuronal cells against oxidative stress and restores

2

rat retinal physiological function from light exposed-damage

3

Fengxia Yan 1,2,#, Haitao Wang1,3,#, Yang Gao4, Jiangping Xu3 and Wenhua Zheng1,2*

4 5 6

1

7

2

8

Sciences, Sun Yat-sen University, Guangzhou, China

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3

Faculty of Health Sciences, University of Macau, Taipa, Macau, China The First Affiliated Hospital and Neuroparmacology, School of Pharmaceutical School of Pharmaceutical Sciences, Southern Medical University, Guangzhou,

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China

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4

Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China

12 13 14 15 16 17 18 19 20 21 22 23 24

*Corresponding author:

25

Prof. Wenhua Zheng, Ph.D, MD.

26

Faculty of Health Sciences, University of Macau

27

Room 4021, Building E12, Avenida de Universidade, Taipa, Macau and the School of

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Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China

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Tel: +853-88224919;

30

Email: [email protected]

31

# Equal contribution as first author

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ACS Paragon Plus Environment

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1

Abstract

2

Oxidative stress plays a key role in the pathogenesis of age-related macular

3

degeneration (AMD), a leading cause of severe visual loss and blindness in the aging

4

population which lacks any effective treatments currently. In this study, artemisinin, a

5

well-known anti-malarial drug was found to suppress hydrogen peroxide

6

(H2O2)-induced cell death in retinal neuronal RGC-5 cells. Artemisinin, in the

7

therapeutically

8

accumulation

9

mitochondrial membrane potential and decreased cell apoptosis in RGC-5 cells

10

induced by H2O2. Western blot analysis showed that artemisinin upregulated the

11

phosphorylation of p38 and extracellular signal–regulated kinases1/2 (ERK1/2) and

12

reversed the inhibitory effect of H2O2 on the phosphorylation of these two kinases.

13

Moreover, protective effect of artemisinin was blocked by the p38 kinase inhibitor

14

PD160316 or ERK1/2 kinase pathway inhibitor PD98059, respectively. In contrast,

15

c-Jun N-terminal kinase inhibitor and rapamycin had no effect in the protective effect

16

of artemisinin. Taken together, these results demonstrated that artemisinin promoted

17

the survival of RGC-5 cells from H2O2 toxicity via the activation of the p38 and

18

ERK1/2

19

concentration-dependently reversed light exposed-damage (a dry AMD animal model)

20

of rat retinal physiological function detected by flash electroretinogram. These results

21

indicate that artemisinin can protect retinal neuronal functions from H2O2-induced

22

damage in vitro and in vivo and suggest the potential application of artemisinin as a

23

new drug in the treatment of retinal disorders like AMD.

24

.

25

Key words: AMD, Artemisin, H2O2, ROS, p38, ERK1/2

relevant of

dosage,

intracellular

pathways.

concentration-dependently

reactive

Interestingly,

oxygen

intravitreous

species

injection

26

2


attenuated (ROS),

of

the

increased

artimisinin,

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1

Introduction

2

Age-related macular degeneration (AMD), a progressive neurodegenerative

3

disease, has become one of the major causes of blindness in the world which affects

4

vision in the elderly people in western countries 1. According to latest data from

5

WHO, about 13.5 million people all over the world suffer severe visual impairment

6

or legal blindness due to AMD 1, and the trend is seen rising in the elderly people.

7

What's worse, along with AMD induced loss of vision, their reading, writing and

8

driving are also seriously affected, which ultimately lead to reduced quality of life2.

9

On the other hand, patients with visual loss from AMD are also accompanied by

10

increased incidence of anxiety and depression3.

11

Despite the severity of the problem, the etiology and pathogenesis of AMD

12

are still unclear, and the clinical treatment available currently is far from satisfactory.

13

AMD consists of wet AMD (about 5%) and dry AMD (more 90%). Current

14

therapeutic strategies available are mainly effective in retinal damage of wet AMD 4,

15

however, there is a lack of effective therapies for the majority of AMD patients (dry

16

AMD). Thus, effective therapeutic drugs for dry AMD are most needed.

17

AMD is a multifactorial disease influenced by various factors like metabolic,

18

genetic, biochemical and environmental factors. Recent studies indicate that oxidative

19

stress is a key factor involved in the pathogenesis of AMD 5-7. Retinal neurons live in

20

a highly oxygen-rich microenvironment which usually generates big quantity of

21

photo-oxidation waste products thereby the neurons are continuously exposed to

22

oxidative

23

autophagy and degradation of shed photoreceptor outer segments which lead to the

24

accumulation of lipofuscin in cell lysosomes8. Lipofuscin, a pro-oxidant compound,

25

produces reactive oxygen species and accumulates in cell lysosomes, which is also

26

thought to reduce phagocytic ability and causes apoptosis of cell9. Oxidative damage

27

may activate various components within the cells and increase the production of

28

cytokines. These pathological changes may contribute to the occurrence and

29

development of AMD

30

oxygen species (ROS) such as hydrogen peroxide lead to imbalance between oxidant

stress.

In

addition,

10,11

.

AMD

is

an

age-related

injury

to

The excessive generation of free radicals and reactive 3



1

system and antioxidant defense system that seem to be the most responsible factor in

2

AMD7,12. Since oxidative stress involves in almost all other assumptive pathogeneses

3

and all risk factors of disease, it may be a key factor for the initiation and progression

4

of the AMD.

5

Artemisinin, a chemical compound of the sesquiterpene lactone isolated from

6

the sweet wormwood plant (Artemisia annua), and its derivates have been clinically

7

used to treat malaria for decades and have saved millions of lives since their

8

application 13,14. Owing to the development of artemisinin for the treatment of malaria,

9

Professor Youyou Tu in China was announced as the 2015 Nobel Prize winner in

10

medicine and physiology. Apart from its potent anti-malarial effect, artemisinin also

11

shows significant anti-inflammatory15, anti-viral16 and anti-cancer properties16.

12

Additionally, artemisinin is cheap and clinically safe with few side effects and should

13

be more suitable for long-term clinical applications like the treatment of AMD

14

patients.

15

In support of above notion, our recent studies showed that artemisinin was able

16

to promote the survival of retinal pigment epithelial cells, cells with critical support

17

functions for photoreceptors are first affected in retinal disorders like AMD, from

18

oxidative insult via the activation of the MAPK/CREB pathway17. Moreover,

19

artemisinin at low concentration was found to induce neurite outgrowth and

20

promotion of neuronal differentiation in PC12 cells via activation of extracellular

21

signal–regulated kinases1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38)

22

signaling pathways 18. These findings indicated the neurogenic effects of artemisinin.

23

However, the effect of artemisinin on the retinal neuronal cells which ultimately

24

degenerate in AMD or other retinal disorders is still elusive.

25

In the present study, we adopted an available model of oxidative stress with

26

hydrogen peroxide (H2O2) in retinal neuronal cells (RGC-5 cells) to investigate the

27

survival promoting effect of artemisinin in retinal neuronal cell from oxidative stress

28

and its relative molecular mechanisms. We found that artemisinin protected RGC-5

29

cells from H2O2-induced injury via modulating the p38/ERK1/2 signal pathways.

30

Flash electroretinogram analysis in rat eye also showed that artemisinin, 4


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1

concentration-dependently reversed light exposed injury of retinal physiological

2

functions in rat. These results indicate that artemisinin is able to protect retinal

3

neuronal cells from oxidative stress and strongly suggest its potential as a candidate

4

drug for the treatment of retinal neurodegenerative diseases like AMD.

5 6

Results

7

Artemisinin protected RGC-5 cells from oxidative damage induced by H2O2.

8

RGC-5 cells were incubated with various doses of H2O2 in 96-well plates and the

9

viability of cells was measured by the MTT assay 24 h later. As shown in Fig.1A,

10

H2O2 promoted the cell growth at low concentrations while induced cell death in a

11

concentration-dependent manner at higher concentrations. H2O2 induced cell death in

12

RGC-5 cells with approximately 60% of the cells left viable at 100µM and this

13

concentration was selected for subsequent studies. To study the effect of artemisinin

14

on H2O2-induced cell death, the MTT assay was used. Fig.1B showed that artemisinin

15

protected RGC-5 cell death induced by H2O2 in a concentration-dependent manner

16

and reached maximal at 100µM.

17

Artemisinin inhibited apoptosis induced by H2O2 in RGC-5 cells. To investigate

18

the effect of artemisinin and H2O2 on the apoptosis of RGC-5 cells, Hoechst and JC-1

19

staining assays were used. As shown in Fig.2 A row 2 and Fig.2B, H2O2 caused

20

nuclear condensation, while artemisinin reversed the effect of H2O2. Moreover, JC-1

21

staining showed that H2O2 decreased the mitochondrial membrane potential (an early

22

apoptotic sign) as shown by the red-green fluorescence ratio (Fig. 2A row 3, colom 2

23

vs 1 and 2C) while pre-incubating the cells with artemisinin resulted in the recovery

24

of the mitochondrial membrane potential (Fig. 2A row 3, colom 3 vs 2 and 2C). To

25

further confirm the involvement of apoptosis, RGC-5 cells were harvested and

26

analyzed by flow cytometry. Fig. 2D showed that the percentage of apoptotic cells in

27

the control group, H2O2 group and H2O2 + ART group was 0.737%, 18.0% and 6.22%

28

respectively. In this experiment, we also found that H2O2 increased the expression

29

level of Bax, while artemisinin attenuated this effect (Fig. 2H). These results

30

indicated that treatment with artemisinin attenuated apoptosis induced by H2O2 in

31

RGC-5 cells.

32

Artemisinin reduced intracellular ROS generation in concentration-dependent 5



1

manner. Oxidative stress is the major cause of H2O2-induced cell death. To study the

2

effect of H2O2 on cellular ROS levels in RGC-5 cells, DCFH-DA staining assay was

3

used. As shown in Fig.2F, H2O2 treatment significantly increased fluorescence

4

intensity of ROS by 98.5% when compared with control group, however, artemisinin

5

decreased the cellular intracellular ROS induced by H2O2 in concentration-dependent

6

manner (Fig.2E).

7

Artemisinin protected RGC-5 cells from H2O2 through the p38/ERK1/2 pathway.

8

Knowing that artemisinin protected RGC-5 cells from injury induced by H2O2, we

9

next investigated the signaling pathways responsible for the protective effect of

10

artemisinin. P38MAPK and ERK1/2 pathways play a critical role in cell survival

11

effects and apoptotic insults

12

ERK1/2 pathways in H2O2-induced cell death. RGC-5 cells were pretreated with

13

PD160316 (10µM, a p38 MAPK inhibitor) or rapamycin (100nM, a mTOR inhibitor)

14

or JNK inhibitor (10µM) or PD98059 (30µM, an ERK inhibitor), and then cells were

15

exposed to 100µM of H2O2 with or without artemisinin for 40 min, and cell viability

16

was determined by MTT assay. As shown in Fig.3A and D, PD160316 (56.67±3.45)

17

or PD98059 (72.23±1.57) significantly blocked the protective effects of artemisinin

18

on H2O2-induced cell death, respectively. However, JNK inhibitor or mTOR inhibitor

19

rapamycin had no effect (Fig.3B and C). These results suggested the involvement of e

20

p38/ERK1/2 pathway in the protective effect of artemisinin.

21

Artemisinin stimulated the phosphorylation of p38 and ERK1/2 in RGC-5 cells

22

and reversed the inhibitory effect of H2O2 on these pathways. Since p38 and

23

ERK1/2 are involved in the effect of artemisinin, we next studied the effect of H2O2

24

and artemisinin on the phosphorylation of p38 and ERK1/2 in RGC-5 cells. First,

25

RGC-5 cells were treated with different concentrations of H2O2 at different time

26

points, and the phosphorylation of p38 and ERK1/2 were determined by Western t

27

blotting. Fig. 4A, showed that high concentrations of H2O2 (100-1000 µM) decreased

28

the phosphorylation of p38 and ERK1/2 in dose- and time-dependent manner (Fig.4A

29

and B) in RGC-5 cells. Subsequently, we studied the effect of artemisinin on

30

phosphorylation of p38 and ERK1/2 in RGC-5. RGC-5 cells were treated with

31

different concentrations of artemisinin for different time points and then P-p38 and

19,20

. We, therefore, tested the role of p38 MAPK and

6


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1

P-ERK1/2 were determined by Western blot. Fig.5D and G showed that artemisinin

2

increased the phosphorylation of p38 and ERK1/2 in concentration and time–

3

dependent manner and reached the maximal effect at 100 µM and 80 min.

4

To investigate the interactions of artemisinin and H2O2 on the phosphorylation of

5

p38 and ERK1/2 in RGC-5 cells, cells were pretreated with different concentrations

6

of artemisinin and then with 100µM H2O2, and the phosphorylation of the two

7

proteins was determined by Western blot. Fig.5A showed that 100µM H2O2 decreased

8

the phosphorylation of p38 and ERK1/2 in RGC-5 cells while artemisinin

9

concentration-dependently reversed the inhibitory effect of H2O2. These results

10

further supported the role of the p38/ERK1/2 pathway in the protective effect of

11

artemisinin.

12

To further confirm the role of p38, ERK1/2, JNK1/2 and mTOR, cells were

13

pretreated with a variety of kinase inhibitors such as PD160316 or PD98059 or JNK

14

inhibitor (10 µM) or Rapamycin before treatment of artemisinin. As shown in Fig.6,

15

artemisinin-induced phosphorylation of P38 or ERK was attenuated by the p38

16

inhibitors PD160316 and specific ERK pathway inhibitor PD98059, respectively, but

17

not by the JNK inhibitor or the mTOR inhibitor rapamycin as shown in Fig.6（A, B ,

18

C, D and M）, but the phosphorylation of JNK1/2 and mTOR had no obvious change

19

in H2O2 after artemisinin treatment Fig.6（E, F）. These results indicated that

20

PD160316 is able to specifically block the phosphorylation of p38 induced by

21

artemisinin while PD98059 is able to block the phosphorylation of ERK induced by

22

artemisinin in our experimental conditions and further confirmed the role of the

23

p38MAPK/ERK pathways in the protective effect of artemisinin.

24

Artemisinin treatment

25

light-induced retinal damage in vivo. Having known that artemisinin plays a

26

protective effect against oxidative stress-induced RGC damage, we then moved to in

27

vivo studies. In the present study, light exposure was used to induce retinal damage,

28

as it has been shown that light exposure increases the generation of ROS, and

29

activates mitochondria-dependent apoptosis in RGC-5 cells. To evaluate the retinal

improved

RGC

and

Entoretina

7


function

after


1

function among groups, flash electroretinogram (FERG) was performed using Roland

2

RETIport visual electric physiological system after light-induced retinal injury. FERG

3

reflects the sum reaction of the total retina alterations in FERG were analyzed. As

4

shown in Fig.7. The A-wave of Rod reaction originates from the reaction of rod cells.

5

Muller cells and on-bipolar cells contribute to B-wave of Rod reaction. The A-wave

6

of Max reaction originates from the reaction of rod and cone cells. The B-wave of

7

Max reaction originates from the reaction of Muller cells and on-bipolar cells, and

8

other cells in the inner retina. OPS wave is originated from the inner retina, and is

9

thought to be the inflection of the inhibitory feedback loop between amacrine cells

10

and bipolar cells. In the light-induced injury group, A-wave amplitudes of Rod

11

reaction(59.81±5.35), B-wave amplitudes of Rod reaction(44.03±4.04), Max reaction

12

A wave(54.09±7.76), Max reaction B wave(49.24±3.52) and OPS amplitude

13

(59.8±4.66) in FERG were significantly decreased compared to the control group.

14

However, The Rod and Max reaction and OPS amplitude in FERG were all highly

15

enhanced in the artemisinin-treated group, compared to the light-induced injury group.

16

These results demonstrated that artemisinin had a protective effect on RGC and the

17

inner retina function after light-induced injury, and the protective effect of artemisinin

18

was dose dependent. A representative of each group in Rod reaction, Max reaction,

19

OPS amplitude, and FVEP amplitude is shown in supplementary Fig.S1.

20 21

Discussion

22

AMD is a neurodegenerative disease and the leading cause of blindness in the elderly

23

people 21. Although the etiology of AMD is still unclear, oxidative stress is considered

24

as one of the most important risk factors. Oxidative stress increases the

25

over-production of reactive oxygen species, leads to the oxidative injury of retina22,23.

26

Recently, clinical research found that antioxidant vitamins and zinc supplements

27

could slow down the progression of AMD disease, but effective treatment is still

28

lacking

29

artemisinin and evaluated its role in H2O2-induced RGC-5 cells damage. Our results

30

showed that H2O2 at low concentrations promote the growth of RGC-5 cell, which

24,25

. In the present study, we investigated the antioxidant activities of

8


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1

might be because low levels of H2O2 can increase the synthesis of cell DNA and

2

induce up-regulation of cell c-fos and c-myc gene expression. These genes are related

3

to the cell proliferation. H2O2 also induced the apoptosis of RGC-5 cell and

4

artemisinin protected RGC-5 cells from H2O2-induced oxidative stress injury via

5

p38/ERK1/2 pathways. To our knowledge, this is the first study exploring the

6

therapeutic effect of artemisinin towards AMD. As the therapeutic strategies for AMD

7

are limited, our study provides a promising alternative for the treatment of this

8

neurodegenerative disease.

9

Artemisinin, a chemical compound, isolated from the sweet wormwood plant

10

(Artemisia annua) has been used as an anti-malarial drug in Chinese pharmacopoeia

11

for a long history26. In the present study, we found that artemisinin decreased

12

H2O2-induced production of ROS, mitochondrial damage and cell apoptosis in

13

RGC-5 cells. These results indicated that artemisinin is able to protect retinal

14

neuronal cells from oxidative stress. This is consistent with our previous report where

15

artemisinin was able to promote the survival of PC12 cells from SNP injury and

16

suggested that artemisinin may be a potential drug for neuronal degenerative

17

disease17. Artemisinin is a FDA approved drug and the dosage used in our study was

18

therapeutically relevant. These findings taken together strongly suggested that

19

artemisinin is safe protectant of retinal neuronal cells with significant clinical

20

potential.

21

To determine the molecular mechanisms behind antioxidative properties of

22

artemisinin, we studied the effects of artemisinin on p38 and ERK1/2 signaling

23

pathways. Our results indicated that H2O2 decreased the phosphorylation of p38 and

24

ERK1/2 in RGC-5 cells while artemisinin dose-dependently increased the

25

phosphorylation of p38 and ERK1/2 and reversed the effect of H2O2. These results

26

suggested the involvement of p38MAPK and ERK in the protective effect of

27

artemisinin. To further confirm the role of p38 and ERK1/2 in antioxidant capacity of

28

artemisinin, p38 inhibitor PD160316 or ERK pathway inhibitor PD98059 was used

29

and the results verified that the effect of artemisinin on both phosphorylation of these

30

kinases and cell survival was blocked by either P38 inhibitor PD160316 or ERK 9



Page 10 of 31

1

pathway inhibitor PD98059. These pharmacological inhibitor studies further

2

confirmed the critical role of these two kinases in the effect of artemisinin against

3

H2O2-induced cell death.

4

The activation of MAPKs by artemisinin is not solely restricted to RGC-5 cells.

5

Sarina et al. had found that artemisinin induced the neurite outgrowth in PC12 cells

6

by activation of p38 MAPK and ERK1/218. Dihydroartemisinin induced apoptosis in

7

HL-60 leukemia via regulation of the p38/ERK1/2pathways, although their regulation

8

is very slow (three hour or later after addition) and the phosphorylation of p38 MAPK

9

but not the ERK1/2 and JNK is required for dihydroartemisinin-induced apoptosis27.

10

Artesunate had also been shown to attenuate LPS induced inflammatory responses in

11

microglial BV2 cells via activation of ERK1/2 and JNK1/228. These findings are

12

consistent with MAPK as a serine/threonine kinase playing key roles in mediating

13

proliferation, differentiation and apoptosis of cells29.

14

Our study firstly showed that the protective role of artemisinin is mediated

15

through ERK1/2 and p38 in RGC-5 cells. This result not only identified the

16

responsible signaling pathway for artemisinin, but also suggested that p38 and

17

ERK1/2 signaling pathways are pivotal in promoting the survival of RGC-5 cells,

18

both of these may be used as a target for the drug development in the future.

19

Mitochondrion plays a key role in a variety of physiological and pathological

20

processes, such as ATP synthesis, ROS production, and cell apoptosis30. The

21

mitochondrial membrane potential(∆Ψm), as an electrochemical gradient, throughout

22

the inner mitochondrial membrane, is a key for maintaining the

23

function

24

responses for the mitochondrial integrity and

25

mitochondrial function32. Many studies showed that H2O2 damaged the electron

26

transport chain, led to the generation of superoxide33, which further induced the

27

dysfunction of mitochondria, and initiated the accumulation of intracellular ROS. In

28

the present study, we found that H2O2 decreased the membrane potential,

29

significantly

30

induced the expression of Bax, while artemisinin reversed these processes. Therefore,

of

the

respiratory

increased

the

physiological

chain in ATP synthesis31, thus, the

accumulation

∆Ψm

it is an important regulator of

of

10


intracellular

ROS

and

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1

artemisinin is capable of protecting against H2O2 –induced cell death. Although, the

2

exact mechanism, which may be involved in modulating ROS levels directly via

3

antioxidation, or indirectly via mediating p38/ERK1/2 signaling pathways remains

4

unclear as previously reported34. Importantly, these in vitro studies were consistent

5

with in vivo studies in animal models. Retinal neuronal cells were sensitive to

6

long-term light exposure, which induced apoptosis and necrosis and the potential

7

mechanisms

8

mitochondria-dependent apoptosis induced by increased ROS production. Our in vivo

9

data showed that treatment with artemisinin reversed light injury in retinal physical

10

functions. These results further supported that the protective effect of artemisinin is

11

worth further investigation for its use as a compound for the maintenance of vision.

12

Together with our previous study showing that artemisinin protects human retinal

13

pigment epithelial cells from hydrogen peroxide-induced oxidative damage 17, the

14

present results offer strong support for the potential development of artemisinin as

15

therapeutic strategy to prevent retinal pigment epithelial cells and neuronal cells

16

death in the process of AMD.

for

this

biological

phenomenon

might

be

via

activated

17

In summary, our results indicated that artemisinin is able to RGC-5 cells against

18

cell death induced by H2O2 via up-regulating the phosphorylation of p38 and ERK1/2

19

and down–regulating the expression of Bax. Since artemisinin is a FDA approved

20

drug which has being used in clinics for decades with very little side effects, it may

21

be very promising for AMD therapy.

22 23

Materials and methods

24

Materials. Artemisinin, H2O2, PD98059, PD169316, Rapamycin, c-Jun N-terminal

25

kinase (JNK) inhibitor were obtained from Calbiochem (La Jolla, CA, USA).

26

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS),

27

penicillin/streptomycin and trypsin were from Invitrogen (Carlsbad, USA).

28

Anti-phospho-ERK1/2,

29

anti-phosphop38 antibodies were purchased from Cell Signaling Technology

Anti-phospho-JNK1/2,

Anti-phospho-mTOR

11


and


Page 12 of 31

1

(Beverly, MA, USA). Anti-ERK1/2, anti-p38 MAPK, anti-Bax, anti -JNK1/2，anti-

2

-mTOR and anti-β-actin antibody and all secondary antibodies conjugated with HRP

3

were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Methyl thiazolyl

4

tetrazolium (MTT), DMSO, Hoechst 33342, Annexin V-FITC/PI, BCA protein assay

5

kit and other standard reagents were obtained from Sigma Chemical (St. Louis, MO,

6

USA). JC-1 dye (Molecular Probes) was from BestBio (Shanghai, China).

7

Cell culture Retinal ganglion cells line RGC-5 was obtained from cell bank, Sun

8

Yat-sen

9

were used for all experiments and cells were grown routinely in high-glucose

10

Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 100µg/ml of

11

streptomycin, 100 units/ml of penicillin and 10% FBS. Cells were incubated at 37°C

12

in a humidified atmosphere of 5% CO2. Cultured media was replaced twice a week

13

with fresh medium as described above. Stock culture was subcultured at 1:5 ratios at

14

a weekly interval.

15

Treatments To study the effect of H2O2 on the viability of RGC-5cells, cells were

16

treated with different concentrations of H2O2 (from 10 to 1000µM) as indicated; To

17

evaluate toxicity of H2O2, cell viability was measured by MTT assay, while the

18

phosphorylation of signaling proteins was determined by Western blot; To explore the

19

protective effect of artemisinin, cells pretreated with artemisinin (6.25-100µM) were

20

treated with/without 100µM H2O2. The viability and the phosphorylation of RGC-5

21

cells were determined by MTT and Western blot; To investigate the effects of

22

different signaling pathways, cells pre-incubated in the presence or absence of

23

PD160316, PD98059, Rapamycin or JNK inhibitor for 40 min were treated with

24

artemisinin for 1h, cells were then exposed with different dose of H2O2 for indicated

25

time points.

26

MTT assay The metabolic activity of the RGC-5 cells was measured by means of

27

MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. RGC-5

28

cells were sub-cultured in 96-well plates at a seeding density of 5×105cells/well. For

University

(Guangzhou,

China).

12


Cell passages 3–6

Page 13 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

assessing the cytotoxic effect of H2O2, RGC-5 cells were incubated with H2O2 (10 to

2

1000 µM) for 24 h. Then, MTT (0.5 mg/ml) was added into each well and continued

3

incubation at 37℃ for 3-4 h. The medium with MTT solution was aspirated from each

4

well, and subsequently, 100µl DMSO solutions were added to dissolve the formazan

5

crystals. The absorbance of each well that is proportional to metabolic activity of

6

cells was measured at 550 nm using a BIO-RAD680 plate reader (Thermo, Walsam,

7

MA, USA). Three independent experiments were performed.

8

Hoechst 33342 staining for apoptosis To study the cell apoptosis, RGC-5 cells

9

growing on 48-well plates were incubated with H2O2 in the absence (control) or

10

presence (treated) of artemisinin for 24 h in 37°C, cells were then infixed with 4 %

11

paraformaldehyde for 10 min at 4°C. Cells were exposed to Hoechst 33342(10 µg/ml)

12

in PBS for10 min at room temperature. The apoptotic rate of cells was then observed

13

by high content screening system (ArrayScanVTI, Thermo Fisher Scientific, USA).

14

Flow cytometry assay RGC-5 cell (5×105 per well) were seeded into 6-well plates.

15

After treatment with H2O2 and artemisinin, cells were harvested and washed twice

16

with ice-cold PBS and suspended in Annexin V binding buffer. The cell supernatant

17

was stained with 5 µl Annexin-V-FITC and 15 µl propidium iodide (PI) solution. The

18

number of apoptotic cells was analyzed using a FCM. Each experiment was repeated

19

three times.

20

Mitochondrial membrane potential assay The changes of mitochondrial membrane

21

potential were measured using JC-1 staining. JC-1 can enter the mitochondrial

22

membrane and accumulates in it. In healthy cells with normal mitochondrial

23

membrane potential, JC-1 aggregates and emits red fluorescence. On the other hand,

24

in apoptotic or unhealthy cells with low mitochondrial membrane potential, JC-1

25

exists in the monomer form, which shows green fluorescence. The ratio of red /green

26

fluorescence was calculated to assess the mitochondrial membrane potential.

27

According to the manufacturer's directions, RGC-5 cells were suspended in fresh

28

medium and incubated with JC-1(5 mg/l) for 20 min at 37°C. Then the cells were 13



1

washed twice with ice-cold PBS and the fluorescence was captured by inverted

2

fluorescence microscopy at 488-nm excitation and 590-nm emission for red, and at

3

540-nm excitation and 527-nm emission for green.

4

Measurement of reactive oxygen species (ROS) Intracellular accumulation of ROS

5

was evaluated using fluorophotometric quantitation. 2,7-dichlorodihydrofluorescein

6

diacetate (DCFH-DA, Sigma–Aldrich) was used as a molecular probe and it is freely

7

permeable to cells. The DCFA-DA is then hydrolyzed to DCFH by intracellular

8

esterase, where it reacts with ROS and converts to DCF, a green fluorescent dye. The

9

cells were culture with H2O2 with or without artemisinin in 96-well plates,

10

twenty-four hours later, each well was loaded with DCFA-DA, protected from light

11

for 30 min, cells were then washed with PBS and the fluorescence was analyzed

12

using a high content screening system (ArrayScanVTI, Thermo FisherScientific,

13

USA) with the excitation wavelength set at 488 nm and the emission wavelength set

14

at 530 nm.

15

Western blotting analysis Western blotting was performed as described previously 35.

16

Briefly, treated cells from different experimental conditions were washed twice with

17

cold phosphate-buffered saline (PBS), lysed in RIPA buffer [50 mM Tris–HCl pH 8.0,

18

150 mM NaCl,1 mM EDTA, 1 % Igepal CA-630, 1 % sodium dodecyl sulfate (SDS),

19

50 mM NaF, 1 mM NaVO3, 5mM phenylmethysulfonyl fluoride, 10 µg/ml leupeptin

20

(Sigma), and 50 µg/ml aprotinin (Sigma)] or 2× sample buffer [ 62.5 mM Tris–HCl

21

pH (6.8), 2 % (w/v) SDS, 10 % glycerol, 50 mM dithiothreitol, 0.1 % (w/v)

22

bromophenol blue]. Protein concentration was determined by BCA protein assay kit

23

according to the manufacturer’s instructions. Samples with equal amounts of protein

24

were resolved on 8% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and

25

transferred electrophoretically onto PVDF membranes. The phosphorylation of p38,

26

and ERK1/2 were detected using their respective phospho-antibodies and visualized

27

using enhanced chemiluminescence kit according to the manufacturer’s instructions.

28

The density of the scanned protein bands was measured with image J analysis 14


Page 14 of 31

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1

software.

2

Intravitreous injection Intravitreous injection was performed as we previously

3

described 36.

4

University, and were maintained under standard laboratory conditions (20–25 °C,

5

40–70% relative humidity and a 12-h light-dark cycle). All rats had free access to

6

food and water throughout the experiments. The study was approved by the Medical

7

Ethics Committee at Sun Yat-Sen University, and the experiments were conducted in

8

compliance with the ARVO Statement on the Use of Animals in Ophthalmic and

9

Vision Research.

Briefly, the rats were obtained from the animal center at Sun Yat-sen

10

The rats were anesthetized with chloral hydrate (7%, 6 ml/kg body weight,

11

intraperitoneal injection). Oxybuprocaine eye drops were used as local anesthesia.

12

Various concentrations of artemisinin (30µg/ml, 100µg/ml and 300µg/ml) were

13

intravitreally injected into the posterior side of the globe in the right eye. Animals in

14

the control group received PBS injections. All injections were performed with

15

Hamilton microsyringes under direct observation with a surgical microscope.

16

Antibiotic eye drops (ofloxacin) were used directly after the surgery to avoid

17

infection.

18

Flash electroretinogram test. Flash electroretinogram test was performed as we

19

previously described36. Fifty-five SD rats (150-180 g) were randomly divided into

20

five groups: the control group; Light injury group (LIG); LIG+ART low-dose (ART-L,

21

30µg/ml) group; LIG+ART mid-dose (ART-M, 100µg/ml) group; LIG+ART

22

high-dose (ART-H, 300µg/ml). All the groups except the control one were continually

23

exposed to bright light with 16,000 lux before the experiments, and then, they were

24

given 1-2 h dark adaptation. Flash electroretinogram (FERG) was performed using

25

Roland RETIport visual electric physiological system after light induced retinal injury.

26

Rats received anesthesia by intraperitoneal injection of 10% chloralhydrate. The

27

cornea was anesthetized with pontocaine (0.5%), a drop of tropicamide was applied

28

before ERG measurement for pupil dilation. The rats were kept on a Ganzfeld 15



1

chamber during the entire ERG procedure. A reference electrode was inserted into the

2

mouth, and two circular silver chloride electrodes, one placed on the center of the

3

cornea and the other in the tail served as reference and ground electrode, respectively.

4

The intensity of light (2.0 cd s/m2) was calibrated and the preamplifier bandwidth was

5

set at 0.3–300 Hz. Rod reaction and OPS amplitude were recorded using a dim white

6

flash through Retiport visual physiological instrument, whereas Max reaction was

7

recorded using a white flash with 0 dB intensity. The experiment was performed

8

under dim red illumination and all measurements were done in triplicates.

9

Statistical analysis The data were presented as mean ± SEM. All Statistical analyses

10

were performed with the SPSS 13.0 program. Multiple comparisons of the data were

11

done by one-way ANOVA with Bonferroni post hoc tests. Statistical significance was

12

defined as p < 0.05.

13 14

Conflict of Interest

15

The authors declare no conflict of interest.

16 17

Acknowledgments

18

This research was financially supported by the Guangdong Provincial Project of

19

Science and Technology (2011B050200005), the National Natural Science

20

Foundation of China (31371088), SRG2015-00004-FHS from University of Macau

21

and the Science and Technology Development Fund (FDCT) of Macao (FDCT

22

021/2015/A1 and FDCT016/2016/A1). We also want to thank Dr. Uma Guar for her

23

excellent proof read of this manuscript.

24 25

Author Contributions:

26 27 28 29

Wenhua Zheng contributed to conceive and design the experiments, collect data and manuscript writing; Fengxia Yan performed the experiments and manuscript writing; Haitao Wang contributed to par of experiment and manuscript preparation; Jiangping Xu contributed significantly to analysis and manuscript preparation.

30 16


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19



1 2

Figure Legends:

3

Fig.1 Protective effects of artemisinin on H2O2-induced cell death in RGC-5 cells. (A)

4

RGC-5 cells were treated with different concentrations of H2O2 for 24 h, and the

5

viability of cells was determined by MTT assay. The data are means ±SD of three

6

independent experiments, #p

Artemisinin Protects Retinal Neuronal Cells against Oxidative Stress

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