Neuroprotective Effects of Propylgallate Against Oxidative Stress in Retinal Ganglion Cells

Background: Diabetic retinopathy is a group of eye diseases which result in damage to the optic nerve and vision loss, it has seriously affect peoples' health. The purpose of this study is to contrast the neuroprotective effects of curcumin, gastrodin, propylgallate, adenosine. At the same time, we preliminarily explore the molecular mechanism of protective drugs. Methods: In this study, we used 500μM H 2 O 2 treated RGC-5 cells to induce a cellular oxidative stress model. We treated this cell model with four drug monomers: Propylgallate, Curcumin, Gastrodin and Adenosine to nd drug monomers with neuroprotective effect. We used apoptosis PCR array to obtain apoptosis related genes regulated by neuroprotective drugs. Results: We found the Propylgallate treated RGC-5 cells had highest survival rate when compared to Curcumin, Gastrodin, Adenosine treated RGC-5 cells.In addition, it had lowest cell cytotoxicity and apoptotic rate when compared to Curcumin, Gastrodin, Adenosine treated RGC-5 cells.Moreover, the expression of ROS in Propylgallate treated RGC-5 cells was lowest when compared to Curcumin, Gastrodin, Adenosine treated RGC-5 cells. We found that Caspase-3, Caspase-8, and Caspase-9 are the main target genes of Propylgallate which can preliminarily explain the neuroprotective mechanism of Propylgallate against apoptosis.. Conclusion: The present study revealed that the propylgallate has best neuroprotective effects, it may provide a promissing drug to prevent and improve the damage of optic nerve. In this article, we also preliminarily expounded the neuroprotective molecular mechanism of Propylgallate.


Background
Diabetic retinopathy is the most common cause of vision loss among people with diabetes and a leading cause of blindness among working-age adults [1,2]. Globally, the number of people with DR will row from 126.6 million in 2010 to 191.0 million by 2030, and we estimate that the number with vision-threatening diabetic retinopathy (VTDR) will increase from 37.3 million to 56.3 million [1] .Recent advances in the eld of neuroprotection indicate that healthy neurons can be protected from injury, and damaged neurons can be rescued from dying by blocking speci c steps in the cell death cascade [3]. These results suggest that it may be feasible to protect healthy cells and rescue damaged cells in diabetic retinopathy, optic neuropathies, and various retinovascular conditions.
Oxidative stress, which can be de ned as an imbalance between the production and removal of reactive oxygen species (ROS), has been implicated in many types of nerve cell death in the central nervous system (CNS) and in the eye [4,5].It is well known that oxidative strss is able to induce many types of cell apoptosis via releasing of the reactive oxygen species (ROS) to increase the expression of caspase-3 and caspase-9, inluding the neurons, cardiomyocytes and nephrocyte [6].The presence of high concentrations of ROS can overwhelm the cell's natural defense mechanisms and activate pathways that lead to programmed cell death [5].There is agreement that certain agents can ameliorate the cell death process in various kinds of injury that involve oxidative stress.
Curcumin is a bright yellow chemical produced by some plants. It is the principal curcuminoid of turmeric (Curcuma longa), a member of the ginger family, Zingiberaceae [7].Gastrodin is a chemical compound which is the glucoside of gastrodigenin. It has been isolated from the orchid Gastrodia elata and from the rhizome of Galeola faberi [8].Propyl gallate (PG), a polyphenolic compound family that is synthesized by the condensation of propanol and gallic acid [9]. Adenosine, a nucleoside derived primarily from the extracellular hydrolysis of adenine nucleotides, is a potent regulator of in ammation [10].These produces have been widely used anti-in ammation [11], anti-cancer [12,13] and other diseases studies [14].In this study, we explored the anti-oxidative stress ability of these four products. We hypothesis that we can get one product that has best anti-oxidative stress ability.Moreover, we also try to explore its potential mechanism whether the product can protect the retinal ganglion cells against oxidative stress by reducing the expression of apoptotic related gene expression.It will provide a new therapy or prevent the optic nerve injury induced by oxidative stress.

Proliferation Assay
The effects of curcumin, gastrodin, propylgallate, adenosine on the proliferation of RGC-5 cells under oxidative stress condition were detected by MTT tests. The RGC-5 cells were seeded in 96-well plates at concentration of 2000 cells/ml. The cells were pretreated with different concentration of Curcumin (10, 20, 40µM), gastrodin (20, 40, 60µM), propylgallate (5, 10, 20 µM), adenosine (10, 20, 40 µM) for 2 hours, then treated with 500µM H 2 O 2 for 24 hours. Cells without any treatment and cells with the treatment of 500µM H 2 O 2 for 24 hours as control. Following, 10µm MTT was added to each well and cultured for 3 hours. Then the medium was removed and 100 µm DMSO was added to each well. Finally, the plates were read immediately on a plate reader at a test wavelength of 490 nm.

Cytotoxicity Assay
The cytotoxicity of curcumin, gastrodin, propylgallate, adenosine were detected by Cytotoxicity LDH Assay Kit following the manufacturer's instructions. The RGC-5 cells were seeded in 96-well plates at concentration of 2000 cells/ml. The cells were pretreated with different concentration of Curcumin (10, 20, 40µM),Gastrodin(20, 40, 60µM ),Propylgallate(5, 10, 20 µM), Adenosine(10, 20, 40 µM) for 2 hours, then treated with 500µM H 2 O 2 for 24 hours.Following, LDH release agent was added, and the cells were incubated for another 30 minutes, and then 50µL of stop solution to each sample well and mix by gentle tapping. Finally, the plates were read immediately on a plate reader at a test wavelength of 490 nm.

Apoptosis assay
The apoptosis levels of the RGC-5 cells treated with different concentration drugs were subsequently analyzed using an Annexin V-uorescein isothiocyanate (FITC) Apoptosis Detection kit according to the manufacturer's instructions. The cells were seeded in 6-well plates at a density of 1x10 5  20.1% respectively, which was obvious higher than the other 3 groups (Fig. 1B).In addition, the apoptosis assay results discovered that the apoptotic rate of 100 µM and 500 µM H 2 O 2 treated RGC-5 cells were 5.47% and 8.53% respectively, which was obvious higher than the other 3 groups (Fig. 1C).Finally, the ROS expression in 100 µM and 500 µM H 2 O 2 treated RGC-5 cells were 51.11% and 62.96% respectively, which was obvious higher than the other 3 groups (Fig. 1D). According to the above results, we used 500 µM H 2 O 2 to induce oxidative stress model of RGC-5 cells for this study.
The protective effects of Curcumin, Gastrodin, Propylgallate, Adenosine on the proliferation of RGC-5 cells under oxidative stress condition Firstly,we examined the effects of Curcumin,Gastrodin,Propylgallate,Adenosine on the proliferation of RGC-5 cells under oxidative stress condition.As Shown in Fig. 2A, the survival rate of 500µM H 2 O 2 treated RGC-5 cells was about 67.1%.The survival rate of 10 and 20µM curcumin treated RGC-5 cells was about 72.2% and 72.2% respectively, which was higher than the control. By comparison, the 40µM of Curcumin was able to signi cantly reduce the survival rate of 500µM H 2 O 2 pretreated RGC-5 cells, which was only about 40.5%.The survival rate of 20, 40, 60µM Gastrodin treated RGC-5 cells was 65.7%, 67.7% and 68.7%, which was no signi cant difference with the control group (Fig. 2B). Interestingly, the survival rate of 5, 10, 20 µM Propylgallate treated RGC-5 cells was 92.3%, 87.7% and 87.9%, which was signi cant higher than the control group (Fig. 2C).Finally, the survival rate of 10, 20, 40µM Adenosine treated RGC-5 cells was 72.3%, 74.6% and 72.9%, which was higher than the control group (Fig. 2D).

The apoptosis related genes expression of RGC-5 cells treated with Propylgallate under oxidative stress condition
Previous experimental results showed that Propylgallate had an signi cant anti-apoptotic effect,in order to obtain the anti-apoptotic molecular mechanism of Propylgallate,we used the Apoptosis PCR array to obtain the apoptosis related genes regulated by Propylgallate.According to the results of the Apoptosis PCR array (Fig. 6A),the RNA expresion of Caspase-3, Caspase-8, and Caspase-9 is in line with the trend of propylgallate(other genes data no show).As shown in Fig. 6B and C, the RNA and protein expression of Caspase-3, Caspase-8, and Caspase-9 in RGC-5 cells treated H2O2 were signi cantly higher than the control of RGC-5 cells.Suggesting the H2O2 treatment increased RGC-5 cell apoptosis.Interestingly, the expression of Caspase-3, Caspase-8, and Caspase-9 in RGC-5 cells treated with propylgallate after H2O2 pretreatment was obviously reduced.These results suggesting propylgallate was able to protect RGC-5 cells against the injury of oxidative stress.

Discussion
Diabetic retinopathy is a group of eye conditions that damage the optic nerve, the health of which is vital for good vision. It is one of the leading causes of blindness for people over the age of 60. It can occur at any age but is more common in older adults. Bilateral blindness will be present in 8.4 million people in 2010, rising to 11.2 million people in 2020, respectively [15].It has seriously affected the people's health and burdened the government healthcare system. In this study, we compared the neuroprotective effect of curcumin, gastrodin, propylgallate, adenosine, and we found the propylgallate has best neuroprotective effect.
In this study,we used H 2 O 2 to induce cellular oxidative stress model in RGC-5 cells, and we found 500µM Gastrodin, Propylgallate, Adenosine were able to relieve the damage induced by oxidative stress in RGC-5 cells. In this study, we found the Propylgallate treated RGC-5 cells had highest survival rate when compared to Curcumin, Gastrodin, Adenosine treated RGC-5 cells. In addition, it had lowest cell cytotoxicity and apoptotic rate when compared to Curcumin, Gastrodin, Adenosine treated RGC-5 cells. Moreover, the expression of ROS in propylgallate treated RGC-5 cells was lowest when compared to Curcumin, Gastrodin, Adenosine treated RGC-5 cells. Furthermore, we also demonstrated that 19. Zhang Y, Handy DE and Loscalzo J. Adenosine-dependent induction of glutathione peroxidase 1 in human primary endothelial cells and protection against oxidative stress. Circulation research 2005; 96: 831-837. Figure 1 Identi cation of cellular oxidative stress model The protective effects of Curcumin, Gastrodin, Propylgallate, Adenosine on the proliferation of RGC-5 cells under oxidative stress condition