Puerarin protects the retinal pigment epithelium (ARPE19) against hypoxia-induced blood-retinal barrier breakdown and Akt/Bcl2-dependent apoptosis

doi:10.21203/rs.3.rs-249980/v1

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Puerarin protects the retinal pigment epithelium (ARPE19) against hypoxia-induced blood-retinal barrier breakdown and Akt/Bcl2-dependent apoptosis

https://doi.org/10.21203/rs.3.rs-249980/v1

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Background: Puerarin, an isoflavonoid, is a neuroprotectant against many ischemic brain injuries. The research purpose was to investigate whether puerarin treatment can inhibit hypoxia-induced apoptosis and barrier breakdown on human retinal pigment epithelium (ARPE-19) cells.

Methods: Treatment with 100 µM of puerarin showed no ARPE-19 cytotoxicity. Treatments with 50 µM or 100 µM of puerarin significantly preserved cell viability under hypoxic conditions for 12 h (p < 0.05). Moreover, treatments with 50 µM and 100 µM of puerarin significantly reduced the proportion of Annexin V- and PI-positive ARPE cells under hypoxic conditions (p < 0.05). The TER analysis and junctional protein staining demonstrated that treatments with 50 µM or 100 µM of puerarin prevented the hypoxia-induced barrier disruption in APRE-19 cultures. In contrast to the untreated group, treatment with puerarin significantly increased the level of Bcl-2 and decreased the levels of p-Bad, Bax, and cleaved caspase 3 in RPE cell under hypoxic conditions (p < 0.05). Treatment with puerarin maintained Akt1 activation in the ARPE-19 cells under hypoxic conditions (p < 0.05), and inhibition of PR-induced Akt1 activation abolished the protective effect of puerarin in ARPE-19 cells under hypoxic conditions.

Conclusion: Our results demonstrated that treatment with puerarin protected RPE cell against hypoxia-induced blood-retinal barrier breakdown and Akt-dependent apoptosis. These findings suggest that puerarin could be developed as an alternative treatment for ischemic and hypoxic retinal injuries.

Ophthalmology

Puerarin

Human retinal pigment epithelium cells

Hypoxia

Apoptosis

PI3K/Akt pathway

The primary cause of blindness among people older than 50 years is reported to be age-related macular degeneration (AMD). Advanced dry AMD is characterized by photoreceptor and retinal pigment epithelium (RPE) loss. (1) Studies have suggested that drusen between the RPE layer and the choriocapillaries may block the oxygen diffusion and nutrients supply from the choriocapillaries to the RPE layer and photoreceptors. (2, 3) Lack of sufficient blood supply to the retina results in hypoxia, tissue ischemia, and the fast defeat of energy generation with subsequent processes of cell death. (4) RPE and photoreceptor cells die gradually under retinal ischemia; therefore, hypoxia and ischemia have been associated with the pathophysiology of retinal degeneration. (4) In wet AMD, hypoxia may induce expression of angiogenic factors, producing choroid neovascularization. (5) In addition, hypoxia is a detrimental factor for the blood–retina barrier (BRB) and leads to the progression of wet AMD. (6) Protecting RPE cells form hypoxia is crucial to prevent or reduce the progression of retinal degenerations.

Puerarin is the primary bioactive ingredient from the Pueraria lobata root, widely known as “Gegen” in traditional Chinese medicine. It possesses a broad spectrum of pharmacological effects such as vasodilation,(7) cardio-protection,(8) neuroprotection,(9) and antioxidant ,(10) anticancerous, (11) and anti-inflammation properties; (12) in addition to inhibiting diabetic ocular complications. (13) Studies have shown that puerarin has neuroprotective effects against toxicities induced by glutamate,(14) N-methyl-D-aspartic acid,(15) ß-amyloid, (16) and 1-methyl-4- phenylpyridinium (MPP+).(17) In vivo have also demonstrated that puerarin is neuro-protectant in rat models of spinal cord (18, 19)and cerebral ischemia injuries (20, 21) through activation of the PI3k/Akt signaling pathway. (9) Thus, puerarin may be an ideal protective candidate for RPE cells against hypoxic stress.

RPE is the pigmented cell layer in the inner sensorineural layer on the Bruch’s membrane. (22) The RPE transports oxygen and nutrients from the choroid to the photoreceptor cells, while also remove wastes of photoreceptors for disposal to the choroid. However, an insufficient supply of nutrients and oxygen from choroidal capillaries result in RPE damage. (23) Remarkably, Akt activation was reported to protect RPE cells under normal conditions and during disease circumstances such as AMD.(24) Moreover, puerarin has been reported to activate the Akt pathway to produce its antiapoptosis effects in neuroblastoma,(17) cardiomyocyte,(25) and pancreatic β cells.(26) Thus, we hypothesized that puerarin might trigger the Akt activation to prevent RPE cell death under hypoxic conditions. We evaluated the possible cytoprotective effects of puerarin in hypoxia-induced RPE cell apoptosis and its possible mechanisms.

1.1 Culture of ARPE-19

The human retinal pigment epithelial cell line, ARPE-19, was expaned in Dulbecco’s modified Eagle’s medium (Gibco; Thermo Fisher Scientific, Grand Island, NY, USA) containing 3 mM L-glutamine (Gibco), 0.5% fetal bovine serum (Gibco), 100 U/mL penicillin G, and 100 g/mL streptomycin sulfate (Gibco) at 37°C in an environment containing 5% CO₂. Cell suspensions were added into 96-well microplates at 5000 cells/well in 100 µL. The ARPE-19 cells were incubated overnight to reach 80% to 90% confluence before further experiments.

1.2 Hypoxic conditions

RPE cells were added into 96-well plates at a density of 5 x 10³cells in each well. After 24h, the culture was washed with phosphate buffered saline (PBS), before adding fresh culture medium to them. For the hypoxia experiment, cells were incubated at 37^˚C in a controlled chamber at 1% O₂ in the presence of 5% CO₂and 94% N₂ (ASTEC, Fukuoka, Japan). Cells were incubated under normal conditions (20% O₂, 5% CO_2, and 75% N₂) served as normoxic controls.

1.3 Cell viability assay

We measured cell viability using WST-8 cell proliferation assay kits (WST-8, Cayman, USA) in triplicate for each group according to the manufacturer’s guideline. We read the OD450 nm in each well to assess cell viability.

1.4 Detection of apoptosis

We detected apoptosis in ARPE-19 by using the Annexin V PI Apoptosis Detection Kit (Genedirex, USA). Briefly, ARPE-19 cells were reacted with Annexin V (1 mg/ml, Genedirex) and propidium iodide (PI) solution (5 mg/ml, Genedirex) for 20 min. An epi-fluorescence microscope (Axioskop; Carl Zeiss Meditech, Thornwood, NY, USA) was used to detect early (Annexin V⁺/PI^-) and late (Annexin V⁺/PI⁺) apoptotic cells. We calculated 6 randomly selected areas of 62,500 μm² in each well and calculated their averages as the mean density of apoptotic cells. All detections were performed in triplicate.

1.5 Measurement of transepithelial electrical resistance

To ensure the development of a properly formed monolayer and tight junctions within the Transwell plates, we measured the transepithelial electrical resistance (TER) using an epithelial volt-ohm meter (World Precision Instruments, Sarasota, FL, USA), after applying the designed treatments to the cultures. To determine TER (Ω/cm²), we subtracted the values of Transwells without cells from the experimentally measured values.

1.6 Immunofluorescence staining of tight junction proteins

We immunostained the ARPE-19 cell monolayer on glass coverslips for tight junction proteins, ZO-1 and Occludin. Briefly, cells were fixed in methanol for 15 min and blocked by 2% bovine serum albumin buffer (Sigma-Aldrich Corp, USA) for 1 hour. Rabbit anti-Occludin and mouse anti-ZO-1 (1: 200 dilution; Abcam, Cambridge, MA, USA) were added and incubated overnight. The next day Alexa 488 secondary antibodies (1:200 dilution; Invitrogen; Thermo Fisher, USA) were added for 1 hour at 25^˚C. Pictures were taken by using a fluorescence microscope (Axioskop; Carl Zeiss Meditech, Thornwood, NY, USA ).

1.7 Western blot analysis

We collected cells after 24 hours under hypoxic conditions in each group. We used Western blots to quantitate levels of pro-apoptosis and antiapoptosis proteins, tight junction proteins, and PI3/Akt phosphorylation, ERK phosphorylation in each group. The 4-12% NuPAGE was used to separate the protein extracts. The gels were transferred onto polyvinylidene difluoride membranes. The membranes were then reacted with cleaved caspase-3, p-Bad, Bax, Bcl-2, p-Akt, p-PI3K, p-ERK, ZO-1, and Occludin antibodies (Cell signaling, USA) primary antibodies at 4 °C. Next, the membranes were reacted with a secondary antibody for 1 hour. Signals were developed by using an enhanced chemiluminescent substrate, and images were acquired by a Western blot analyzer. We calculated the relative density using the ImageJ software (https://imagej.noh.gov/ij/). In brief, the mean gray value was measured to quantify the protein band in the quantification software. For band detection, the rectangle tool was used to select a region of interest. For band quantification, the area under the curve of the detected band was used to represent the band density. The final relative density is the ratio of protein of interest to internal loading control.

1.8 Statistical analysis

All statistical analyses were performed using a GraphPad Prism. Data are presented as means ± standard deviations. We applied Mann–Whitney U-tests for comparisons between groups and considered P < 0.05 as statistically significant.

2.1 Cytotoxicity of puerarin in the ARPE-19 cell

We examined puerarin cytotoxicity by incubating ARPE-19 cells with the designed-concentrations of puerarin (10, 100, 200, 500, and 1000 µM) for 24 hours, and using WST-8 assays to determine their viability. The viability of RPE cells was significantly reduced by treatment with 500 and 1000 µM puerarin compared to the viability of untreated-RPE cells (Figure 1A; 500 µM puerarin, P = 0.029; 1000 µM puerarin, P = 0.0006). To measure long-term puerarin effects on cytotoxicity, we treated the ARPE-19 cells with puerarin at 10, 100, and 200 µM for 1, 2, 5, and 7 days. The ARPE-19 cells treated with 200 µM of puerarin showed lower viability at 2, 5, and 7 days posttreatment (Figure 1B, p < 0.0001). Puerarin concentrations lower than 100 µM resulted in no cytotoxicity in the ARPE-19 cells during 7 days in culture.

2.2 Puerarin reduced the hypoxia-induced apoptosis in ARPE-19 cells

RPE cell death progressed in a time-dependent manner under hypoxic culture conditions (Figure 2A). We observed a dramatic decrease in ARPE-19 cell viability 8 hours after initiating the incubation in the anaerobic chamber (Figure 2A, P = 0.0001). Puerarin at 50 and 100 µM inhibited the hypoxia-induced RPE cell death significantly. However, puerarin at 10 µM had no significantly protective effect on ARPE-19 cells after the same hypoxic incubation period (Figure 2A, P = 0.0001). Next, we examined whether the protective effect is involved in an antiapoptotic response. The Annexin V staining demonstrated that puerarin treatment at 50 and 100 µM reduced the proportion of Annexin V-positive and PI-positive cells under hypoxic conditions (Figure 2B). The proportions of Annexin V-positive cells in the hypoxic control, the 10 µM PR-treated, the 50 µM PR-treated, and the 100 µM PR-treated cells were 55.3 ± 5.5%, 50.7± 7.0%, 14.6 ± 4.5%, and 12.8 ± 3.9%, respectively. The proportions of PI-positive cells in the hypoxic control, the 10-µM PR-treated, the 50-µM PR-treated, and the 100-µM PR-treated cells were 72.5± 6.6%, 63.7± 4.0%, 21.7± 9.4%, and 21.0± 5.9%, respectively (Figure 2B). Treatment with 50 µM puerarin reduced the percentage of Annexin V-positive cells 40.7% (P = 0.0004) and that of PI-positive cells 50.8% (P = 0.0004) compared to the percentages in the hypoxic control group. Treatment with 100 µM puerarin reduced the percentage of Annexin V-positive cells 42.5% (P = 0.0003) and that of PI-positive cells 51.5% (P = 0.0002) compared to the percentages in the hypoxia control cells. These results indicate that treatments with 50 or 100µM of puerarin provided antiapoptotic capabilities in ARPE-19 cells against hypoxic conditions.

2.3 Puerarin inhibited the hypoxia-induced barrier disruption in ARPE-19 cells

The TER of ARPE-19 cell was measured under normoxic and hypoxic conditions and after treatment with or without puerarin (50 µM or 100 µM). The TERs of ARPE-19 cells under normoxic, hypoxic without puerarin, hypoxic with puerarin (50 µM), and hypoxic with puerarin (100 µM) conditions were 161.1 ± 9.6 Ω cm², 66.1 ± 3.5 Ω cm², 127.1 ± 7.2 Ω cm², and 126.4 ± 7.3 Ω cm², respectively. Treatment with 50 and 100 µM of puerarin significantly inhibited the hypoxia-induced TER decreases by 1.92-fold (p < 0.0001) and by 1.91-fold (p < 0.0001), respectively, compared to the TER decrease in the hypoxia without puerarin group (Figure 3A).

To confirm the TER results, we quantitated the tight junctional proteins ZO-1 and Occludin by immunocytochemistry and Western blotting analysis. The expression of levels of ZO-1 and Occludin were significantly reduced 2.5-fold (P = 0.0186) and 1.86-fold (P = 0.0002) in ARPE-19 cells under hypoxia conditions compared to those under normoxic conditions. Treatment with 50 µM of puerarin significantly increased the protein levels of ZO-1 1.99-fold (p = 0.0148) and those of Occludin 1.45-fold (p = 0.0116) in ARPE-19 cells under hypoxic conditions compared to the levels in cells without puerarin treatment. Treatment with 100 µM puerarin in ARPE-19 cells under hypoxia also significantly increased the levels of ZO-1 and Occludin 1.99-fold (p = 0.018) and 1.41-fold (p = 0.02), respectively, compared to the levels in cells without puerarin treatment (Figure 3B).

Treatment with 50 µM and 100 µM puerarin prevented the disorganization of ZO-1 and Occludin induced by hypoxia (Figure 3C). These results indicate that puerarin inhibits hypoxia-induced disruption of the barrier function in ARPE-19 cells.

2.4 Puerarin reduced the levels of apoptosis-associated factors

To elucidate the potential mechanism by puerarin attenuating hypoxia-induced apoptosis, we performed a Western blot analysis to determine a set of apoptosis-associated factors, including cleaved caspase 3, p-Bad, Bax, and Bcl-2. We incubated ARPE-19 cells with 50 and 100 µM puerarin and exposed them to hypoxia for 24 hours before quantitating the proteins.

The levels of p-Bad, Bax, and cleaved caspase 3 were increased by 18-fold (P < 0.0001), 1.37-fold (P = 0.0368), and 15.24-fold (P < 0.0001), respectively, in the hypoxia groups compared with the levels in the normoxia group (Figure 4). The level of Bcl-2 was decreased by 2.24-fold (P = 0.0004) in the hypoxia group compared with that in the normoxia group (Figure 4). Puerarin (50 µM) treatment reduced the levels of p-Bad, Bax, and cleaved caspase 3 by 1.57-fold (P = 0.0022), 1.36-fold (P = 0.0432), and 1.81-fold (P = 0.0003), respectively, compared with the levels without puerarin treatment under hypoxic conditions. Puerarin (100 µM) treatment reduced the levels of p-Bad, Bax, and cleaved caspase 3 by 2.05-fold (P = 0.0002), 1.43-fold (P = 0.0214), and 1.96-fold (P = 0.0002), respectively, compared with the levels without puerarin treatment under hypoxic conditions. The level of Bcl-2 was increased 2.1-fold (P = 0.0009) in the cells treated with puerarin (50 µM) and 2.16-fold (P = 0.0006) in the cells treated with puerarin (100 µM) than in the cells under hypoxia without puerarin (Figure 4). These observations imply that puerarin induced the level of Bcl-2, reduced the levels of Bax and Bad, cleavage of caspase 3, and exhibited antiapoptotic properties through the mitochondrial apoptotic pathway.

2.5 Puerarin induced Akt activation and reduced Akt1-dependent apoptosis

PI3K/Akt (27) and MAPK/ERK (28) pathways regulate cell proliferation and survival. In this study, we examined the pathways of PI3K/Akt and MAPK/ERK. The ARPE-19 cells were exposed to hypoxic conditions for 24 h. Our results showed that hypoxic conditions significantly induced the expression of p-Akt1 1.46-fold (P = 0.0019) and that of p-ERK 1.235-fold (P = 0.023) with a maximal effect after 3 hours compared with expression levels under normoxic conditions. However, the level of p-Akt1 significantly decreased after 12 hours of exposure to hypoxia compared to normocia (Figure 5A, P < 0.0001). The level of pERK showed no statistical difference among each group (Figure 5A).

In order to elucidate whether the Akt inhibitor LY 294002 had an effect on PR-induced PI3K/Akt1 activation, we incubated the ARPE-19 cells with puerarin plus LY 294002 (20 µM) under hypoxic conditions for 24 hours. Our results revealed that puerarin treatment increased the phosphorylation of PI3K 1.66-fold (P = 0.0015), and that of p-Akt1 7.99-fold (P < 0.0001) under hypoxic conditions, compared with the phosphorylation in cells without treatment under hypoxic conditions. However, inhibition of p-Akt by LY 294002 20µM abrogated this effect (Figure 7B, P = 0.365).

Puerarin treatment activated the PI3K/Akt signaling pathway in ARPE-19 cells under hypoxic conditon, and activation of the PI3K/Akt pathway is crucial for the regulation of apoptosis. We further examined the PI3K/Akt1-dependent apoptosis for puerarin treatment. Figure 5D shows the results of Annexin V-FITC/PI stainings to detect apoptotic cells. The percentages of Annexin V-positive cells were 50.87± 6.64 %, 18.33± 3.81%, and 54.33± 7.28%, and those of PI-positive cells were 56.12± 4.01 %, 18.33± 6.29%, and 53.41± 4.5%, respectively in cells without puerarin treatment, cells treated with puerarin (50 µM), and cells treated with puerarin (50 µM) combined with LY294002 (20 µM). We found no significant differences between cells treated with puerarin (50 µM) combined with LY294002 (20 µM) and those without treatments under hypoxic conditions (p = 0.8819 for Annexin V-positive cells and p = 0.7762 for PI-positive cells). The difference between cells treated with puerarin (50 µM) and those treated with puerarin (50 µM) combined with LY294002 (20 µM) was statistically significant (p = 0.0011 for Annexin V-positive cells and p < 0.0001 for PI-positive cells). According to these results, puerarin treatment reduced the hypoxia-induced RPE apoptosis by activating Akt signaling pathway. Besides, inhibiting the Akt activation by LY294002 removed the protective effects of puerarin against apoptosis in hypoxia-induced ARPE-19 cells, suggesting the puerarin antiapoptotic properties on hypoxic RPE cells are dependent on PI3K/Akt signaling.

Hypoxia-ischemia underlies various blinding ocular conditions such as diabetic retinopathy, AMD, and retinal detachment. In addition, these problems are collectively referred to as ischemic retinopathies.(29) The ischemic retinal injury occurs through a self-reinforcing damaging mechanism involving calcium influx, oxidative stresses resulted from hypoxia and energy failure, decrease in mitochondrial enzyme activity, and increase glutamatergic stimulation.(30) A strategy against hypoxia-induced retinal cell damage could restore the healthy environment of the retina and avoid retinal injury. Our results demonstrated that puerarin protected RPE cells against hypoxia-induced cell damages. We found that puerarin treatment regulated Bcl-2/Bax/caspase-3 signaling pathway to prevent hypoxia-induced apoptosis in ARPE-19 cells. In addition, puerarin maintained Akt1 activation to keep survial of ARPE-19 cell under hypoxic condition. Moreover, Puerarin reduced hypoxia-induced blood-retinal barrier breakdown. Thus, our findings suggested that puerarin may be a potential drug for AMD and ischemic retinopathy.

Puerarin has shown strong neuroprotective effects during ischemia/reperfusion, protecting neurons by activating antiapoptotic mechanisms .(20, 31, 32) Thus, we believe puerarin may be a promising candidate to treat retinal degenerative diseases. However, there is no study to investigate the role of puerarin in hypoxia induced apoptotic damage on RPE cells. In this study, we demonstrated that puerarin played a protective role against hypoxia-induced ARPE-19 apoptosis via the Akt1 activation and modulation of Bcl-2/Bax/caspase-3 signaling pathway. These findings suggest that puerarin displayed the antiapoptotic capability through the mitochondrial apoptotic pathway. Mitochondria is a critical element in regulating apoptosis. Antiapoptotic Bcl-2-associated proteins suppressed the activity of Bax.(33) Apoptosis was reported to be suppressed by puerarin treatment in various cell types via regulation of the Bcl-2 family proteins .(9, 34) Taken together, we suggested that puerarin provide the anti-apoptotic activity by modulating Bcl-2, Bax, and Bad in ARPE-19 cells under hypoxic condition.

The inner BRB directly regulates the flux into the inner retina to maintain the homeostasis of the retina.(23, 35) The well-developed tight junctions are crucial for the regulation of fluid and solutes that cross the BRB. In this study, treatment with puerarin kept the TER value in ARPE-19 cells under hypoxic condition. This effect was involved in the regulation of tight junction proteins. Many proteins were reported to be associated with tight junctions. Among them, Occludin and ZO-1 are the well-studied in many cells.(36) In the present study, we found that hypoxia exposure decreased the protein levels of ZO-1 and Occludin in ARPE-19 cells. Hypoxia-induced disorganization of ZO-1 and Occludin were preserved by puerarin treatment. Intereastingly, we also found that treatment with puerarin indcued Akt1 activation in the ARPE-19 cells under hypoxic condtion. In addition, the Akt1 activation prevented the RPE disruption in the hypoxic condition. The previous report demonstrated that erythropoietin-induced Akt activation preserved RPE barrier function in the APRE-19 cells to prevent RPE disruption.(37) Thus, we considered that puerarin-induced Akt1 activation might be involved in RPE barrier function to maintain the organization of junctional proteins in the ARPE-19 cells.

The PI3K/Akt pathway can regulate cell proliferation, differentiation, and survival. This pathway is activated by some growth factors and specific extracellular signals.(27) The PI3K/Akt pathway was reported that its activation is involved in the protection of ARPE-19 cells from apoptosis.(38–40) Our data demonstrated that puerarin triggerd Akt1-dependent cell survial in APRE-19 cells in response to hypoxic stress. The Akt1 activation can regulate cell-survival signals through the Bcl-2 family.(41) Therefore, we suggested that puerarin regulated Akt1/Bcl-2 pathway in response to hypoxia for RPE survial.

In summary, our results suggest that treatment with puerarin prevents the hypoxia-induced apoptosis in ARPE-19 cells via modulation of Akt/Bcl2 pathway. In addition, treatment with puerarin inhibited the hypoxia-induced disruption of BRB. These results indicate that puerarin may be developed as an alternative treatment for ischemic and hypoxic retinal injuries.

AMD

age-related macular degeneration

Puerarin

RPE

retinal pigment epithelium

PBS

phosphate buffered saline

TER

transepithelial electrical resistance

BRB

blood retinal barrier

Bcl-2

B-cell lymphoma 2

ZO-1

Zonula occludens-1

Bax

Bcl-2 associated X-protein

Ethical Approval and Consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of supporting data

Raw data can be obtained from corresponding authors.

Competing interests

The authors declare no competing interests

Funding

This study was supported by a Ministry of Science and Technology Grant from the Taiwanese Government (MOST 103-2314-B-303-007-MY3).

Author contributions

Y-T W, M-A-N-N-L and R-K T designed the experiments. M-A-N-N-L performed the experiment. M-A-N-N-L, Y-T W and D-T-T-T analyzed the data.R-K T contributed the reagents/materials/ analysis tools. M-A-N-N-L, Y-T W and R-K Tsai wrote the manuscript. All contributing authors have read and approved the final version of the manuscript.

Acknowledgments

The authors thank I-Ping Tsai and Yi-Ling Zhang for their assistance with experimental handling and image preparation. This manuscript was edited by Wallace Academic Editing. This study was supported by a Ministry of Science and Technology Grant from the Taiwanese Government (MOST 103-2314-B-303-007-MY3).

Author information

Affiliations:

Institute of Eye Research, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan.

Yao-Tseng Wen, Minh-Anh Nguyen Ngo Le, and Rong-Kung Tsai

Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan.

Rong-Kung Tsai

Faculty of Traditional Medicine, Univeristy of Medicine and Pharmacy at Ho Chi Minh City, Viet Nam.

Minh-Anh Nguyen Ngo Le, Dieu-Thuong Thi Trinh

Corresponding author:

Correspondence to Rong-Kung Tsai

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Puerarin protects the retinal pigment epithelium (ARPE19) against hypoxia-induced blood-retinal barrier breakdown and Akt/Bcl2-dependent apoptosis

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