Neuroprotective Effects of Idebenone on hydrogen peroxide (H2O2)-induced Oxidative Damage in Retinal ganglion cell-5 (RGC-5) Cells CURRENT STATUS: POSTED

Purposes To investigated the neuroprotective effect of Idebenone against H2O2-induced oxidative damage in RGC-5 cells. Methods RGC-5 cells were treated with different concentrations (5, 10, 20μM) of idebenone for 12h prior to addition of 300µM H2O2 for 12 h. The apoptosis of RGC-5 cells were detected by flow cytometry. The changes of mitochondrial membrane potential were detected by JC-1 staining. The autophagy in RGC-5 cells was observed by transmission electron microscopy, and the expression level of autophagy-related protein light chain3, Beclin-1 and mitochondrial membrane potential-related protein Cyt-c in RGC-5 cells were measured by Western blot analysis. Flow cytometry showed that the apoptosis rates in control group, H2O2 group and H2O2-treatment with Idebenone pretreatment groups were (6.48±0.55)%, (27.34±0.51)%, (22.88±0.52)%, (15.45±0.81)%, (12.59±0.58)%, respectively(F = 559.7, P <0.0001). After incubation with H2O2, the number of autophagosomes increased significantly, while which was decreased in H2O2-treatment with Idebenone pretreatment groups. After incubation of RGC-5 cells with H2O2, the mitochondrial membrane potential was significantly decreased, while idebenone could prevent the decrease of MMP. Contrast with control group, LC3 II /I, the expression levels of Beclin-1 and Cyt-c in H2O2 group increased significantly(P<0.05); while contrast with H2O2 group, LC3 II/I, the expression of Beclin-1 and Cyt-c in H2O2-treatment with Idebenone pretreatment groups was significantly decreased(P<0.05). through improving antioxidant capacity, reducing mitochondrial membrane potential decline and the activity of autophagy. In this study, we use exogenous H 2 O 2 to break the balance of oxidation and antioxidation of the cell, to explore the effect of idebenone on hydrogen peroxide-induced autophagy and to explore whether idebenone has antioxidant and protective effects on RGC-5 cells. LC3 a marker protein for autophagy. When autophagy is activated, LC3-I is reacted with phosphorus under the action of ubiquitin-like enzyme Lipoethanolamine coupling to produce LC3-II. LC3-I is located in the cytoplasm, while LC3-II is located in the inner and outer membranes of the autophagosome. And

Recently, researchers discover that oxidative stress is also a key injury factor in GON [2] . Under normal circumstances, reactive oxygen species(ROS) play roles as signaling molecules in vivo. But oxidative stress occurs once the increased ROS content beyond clearance ranges of the body. The imbalance between oxidation and antioxidant capacity is considered to be an important feature of early retinal damage and glaucoma pathology [3] .According to research, H 2 O 2 can induce apoptosis in RGC-5 cells in vitro. Cyt-c is not only a substance transfer by breath Chain electron transfer, but also the principal protein of regulating apoptosis [4] . Mitochondria is the main source of ROS and also the attack target of ROS [4] , which is an important factor in the injury of glaucoma optic nerve [6][7][8][9] . In addition, increased ROS can lead to the decline of MMP, mitochondrial dysfunction occurs, then Cyt-c released from mitochondria lead to apoptosis [6][7][8][9] . Mitochondrial damage will in turn leads to more reactive oxygen species and aggravates oxidative stress. Therefore, mitochondria are important factors in determining cell survival and apoptosis, and play an essential role in glaucomatous optic nerve injury [8,9] .
In addition to the mitochondrial pathway of the apoptotoic pathway, autophagy is also has an impact on the survival of RGCs [11] . Autophagy is a rather conserved lysosomal pathway which is highly regulated by complex factors, such as hypoxia, hunger,some specific medicine. A recent study found that ROS can also mediate autophagy [9] . Autophagy plays a major role in the renewal of cell components and the maintenance of intracellular rings and the stability of the environment [12] . Under normal circumstances, autophagy plays a key role in maintaining cellular homeostasis. However, some recent experimental studies point out that the autophagy is associated with death. Autophagy may be another form of cell death [13] .
Idebenone is an analog of coenzyme Q10, which may be a promising therapeutic strategy for ameliorating glutamate excitotoxicity and oxidative stress in glaucomatous neurodegeneration [14] . It has been proved that coenzyme Q10 can treat mitochondrial disease [15] . We aim to explore whether Idbenone has the antioxidant damage and protective effects on RGCs, and provide some references for therapeutic intervention in glaucomatous optic nerve injury.

Cell line culture
The RGC-5 cells (bought from Fudan IBS) is a mouse retinal ganglion progenitor cell line. In our study, RGC-5 cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin and 100g/mL streptomycin. And cells were cultured at 37℃ in an incubator with 5% CO 2 . The medium was altered every other day. When the cells grow about 80% confluent, they will be passaged at a ratio of 1:4 using 0.25% Trypsin. Usually, the period of passage is two or three days.

5.Transmission electron microscopy(TEM)
The intracellular autophagosomes and autolysosomes were observed by TEM. The cells were washed three times in PBS and fixed with 2.5% PBS buffered glutaraldehyde for 1 hour at 4 °C, then 1% OSO 4 was dehydrated again in the gradient ethanol series for 1 hour and placed flat in Araldite. Ultrathin sections (1µM, thickness) were cut with uranyl acetate and lead citrate and double stained. At last, ultrathin sections were observed using the TEM(JEOL-1230, Tokyo, Japan).

6.Western Blotting Assay
Firstly, cells at a density of 2×10 5 cells/mL were plated onto 100-mm plates and incubated at 37℃ for

Idebenone attenuates autophagy induced by H 2 O 2
Autophagy in each group of cells were observed by transmission electron microscopy (Fig.4). A small amount of autophagosomes and autophagosomes were observed in the normal control group.   In this study, we use exogenous H 2 O 2 to break the balance of oxidation and antioxidation of the cell, to explore the effect of idebenone on hydrogen peroxide-induced autophagy and to explore whether idebenone has antioxidant and protective effects on RGC-5 cells. LC3 a marker protein for autophagy. When autophagy is activated, LC3-I is reacted with phosphorus under the action of ubiquitin-like enzyme Lipoethanolamine coupling to produce LC3-II. LC3-I is located in the cytoplasm, while LC3-II is located in the inner and outer membranes of the autophagosome. And the lipidated form of LC3 (LC3-II) can be considered as the autophagosome marker protein [16,17] . In addition, autophagy related protein Beclin-1 also plays a critical role in the progress of autophagy [18] .
For instance, the discovery of Beclin-1's interacting partners has resulted in the identification of Bcl-2 as a central regulator of autophagy and apoptosis [19] . The expression of LC3-II/I and Beclin-1 can reflect the activity of autophagy.
Idebenone has antioxidant ability to prevent lipid peroxidation and ROS production in multiple systems [19] , such as brain, brown fat.etc. It's waiting to study whether idebenone has the same protective effects in oxidative damage on retinal ganglion cells. Our experimental results suggest that Idebenone can reduce the apoptosis of RGC-5 cells caused by hydrogen peroxide; H 2 O 2 can reduce the MMP and lead to apoptosis and autophagy of RGC-5 while idbenone can prevent the decline of MMP and reduce the activity of autophagy. Our study indicate that idbenone has antioxidant and protective effects on RGC-5 cells. But we must emphasize even under normal circumstances, apoptosis and autophagy exist all the time. According to the experimental research on autophagy in recent years, the amount of autophagy has different effects on the survival number of cells. However, the concrete mechanism of autophagy that induced by H 2 O 2 in RGCs has not been elucidated. Researches has shown that autophagy could regulate biological processes by mediating the related signaling protein degradation [25,26] . Apoptosis and autophagy are not completely independent, they interaction each other in many identical molecular adjustment mechanisms [19,27] . According to the results of our study, maybe, we can prevent or mitigate oxidative damage from two aspects, antioxidant such as maintenance of retinal ganglion cell mitochondrial functions and control the level of autophagy. It may can provide a promising new idea for the optic nerve protection of glaucoma. Figure 1 The results of apoptosis detected by Flow Cytometry. Each chart consists of four areas: Q1 region represents those necrotic cells; Q2 region represents the late stage apoptosis; Q3 region represents the early stage apoptosis; Q4 region represents viable cells. Q2 region adds Q3 region represents the total apoptosis of cells. The numbers in each region represent the proportion of cells in the region of total cells.