PARTⅠ:Novel Protective Effects Of Cistanche Tubulosa Extract Against Low-Luminance Blue Light-Induced Degenerative Retinopathy
Mar 04, 2022
Contact: Audrey Hu audrey.hu@wecistanche.com
Man-Ru Wu Cheng-Hui Lin Jau-Der Hob George Hsiao, Yu-Wen Cheng
School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Department of Ophthalmology, Taipei Medical University, Taipei, Department of Pharmacology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Ph.D. Program in Biotechnology Research and Development, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
Abstract
Background/Aims: Blue light-emitting diode light (BLL)-induced phototoxicity plays an important role in ocular diseases and causes retinal degeneration and apoptosis in human retinal pigment epithelial (RPE) cells. Cistanche tubulosa extract (CTE) is a traditional Chinese medicine with many beneficial protective properties; however, few studies have examined the ocular protective roles of CTE. In this study, we investigated the mechanisms underlying the effects of CTE on BLL-induced apoptosis in vitro and in vivo. Methods: RPE cells were applied in the current in vitro study and cell viability was determined by a 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis-related protein expression was determined by western blot analysis and immunofluorescence staining. Brown Norway rats were used to examine exposure to commercially available BLL in vivo. Hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and western blot assays were used to examine retinal morphological deformation. Results: CTE significantly inhibited hydrogen peroxide-, tertbutyl hydroperoxide-, sodium azide-, and BLL-induced RPE damage. Further, CTE reduced the expression of apoptotic markers such as cleaved caspase-3 and TUNEL staining after BLL exposure by inactivating apoptotic pathways, as shown via immunofluorescent staining. In addition, CTE inhibited the BLL-induced phosphorylation of c-Jun N-terminal kinase, extra signal-related kinases 1/2, and p38 in RPE cells. In vivo, the oral administration of CTE rescued 60-day periodic BLL exposure-induced decrements in retinal thickness and reduced the number of TUNEL-positive cells in the brown Norway rat model. Conclusion: CTE is a potential prophylactic agent against BLL-induced phototoxicity
Introduction
Studies of age-related macular degeneration (AMD) have reported that 8.7% of the world’s population suffers from this disease [1]. AMD is a neurodegenerative disease that causes photoreceptor deformation, retinal pigment epithelial (RPE) atrophy, ganglion cell apoptosis, and central vision loss in the elderly population, and is becoming a global social burden. AMD is classified into either a dry or wet form [2, 3]. Improvements in technology have resulted in people spending more time using modern digital devices, and consequently, people are exposed to blue light (BL) emissions over long periods. However, the issue of BL-induced phototoxicity in the retina is not well-investigated. Overexposure to short-wavelength BL (450–495 nm) results in the generation of reactive oxygen species (ROS), such as superoxide and hydroxyl radicals, which cause increases in oxygen consumption and induce mitochondrial DNA damage. The accumulation of ROS results in oxidative stress and the extensive accumulation of retinoid adducts, which damage the retina further [4]. Research has shown that oxidative stress is a leading factor in the pathogenesis of dry AMD [5]. Furthermore, BL irradiation decreases the expression of retinal antioxidant enzymes, such as superoxide dismutase and catalase, by mediating Bax/Bcl-2 protein interactions and promoting endogenous ROS production. As such, RPE cells and the outer segment mitochondria of photoreceptors suffer from BL irradiation-induced injury [6, 7]. In addition to ROS generation, BL irradiation activates mitogen-activated protein kinases (MAPKs), which play an important role in cell survival and apoptosis. mitochondria-dependent apoptosis-related pro-apoptotic proteins, such as Bax and caspase-3, are activated by BL-emitting diode light (BLL) irradiation via the c-Jun N-terminal kinase (JNK) and p38 pathways in RPE and retinal ganglion cells [8]. Moreover, the phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent anti-oxidative pathways are components of the defense system of RPE cells [9-12].
Cistanche tubulosa extract (CTE) contains phenylethanoid glycoside derivatives and has been used as a traditional Chinese medicine for decades. Echinacoside, acteoside, and isoacteoside are three major active compounds of CTE that are characterized as having neuroprotective, memory-enhancing, immune-regulating, and anti-liver fibrosis properties [13, 14]. Echinacoside is a ROS scavenger and protects against 1-methyl-4-phenylpyridinium ion (MPP+)-induced apoptosis in animal models of Parkinson’s disease [15, 16]. Acteoside protects against amyloid-β-induced neurotoxicity and reduces lipopolysaccharide-stimulated inflammation, attributable to the translocation of Nrf2 from the cytoplasm to the nucleus and binding to antioxidant response elements. The current available therapies for wet AMD, such as pegaptanib, ranibizumab, bevacizumab, and aflibercept, are used to inhibit excessive angiogenesis [17, 18]. However, no effective treatments exist for dry AMD, owing to the multiple pathological complications of this disease.
In this study, we demonstrate the protective effects of CTE in RPE cells and Brown Norway (BN) rats. In addition, we applied long-term low luminance BLL exposure to both in vitro and in vivo models to evaluate the protective effects of CTE. We found that CTE reduced the expression of apoptosis-related proteins after exposure to BLL. Moreover, CTE reduced the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)- positive cells in the retina of BN rats after long-term exposure to low luminance BLL. This study provides further insights into the novel anti-apoptotic effects of CTE and information regarding a new strategy for the treatment of retinal degeneration.
Cistanche tubulosa extract (CTE): phenylethanoid glycoside
Materials and Methods
Chemicals and products
CTE was generously provided by Sinphar Pharmaceutical Company (Yilan, Taiwan). In situ cell death detection kits (Cat. No. 11-684-817-910) were purchased from Roche (Mannheim, Germany).
RPE cell culture
RPE cells (ARPE-19 and ATCC® CRL-2302™) were purchased from the American Type Culture Collection (Manassas, VA) and cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 at 37°C. ARPE-19 cells at passage 19–30 were maintained on 10-cm cell culture dishes (Orange Scientific, Braine-l’Alleud, Belgium). Upon passaging with 0.05% trypsin-ethylenediamine tetraacetic acid (Gibco), the cells were replated at a 1:4 ratio. Before testing, ARPE-19 cells were seeded onto 48-well plates (Orange Scientific) for cell viability analyses at a density of 1.0 ×105 cells/mL and onto 6-cm dishes (Orange Scientific) for western blotting analyses at a density of 2.0 ×105 cells/mL for 24 h. After ARPE-19 cells reached 80% confluency, the following experiments were performed.
Drug treatment and cell viability assay
RPE cells at passage 19–30 were seeded onto 48-well plates and co-treated for 24 h with different concentrations of CTE, hydrogen peroxide (H2O2), tert-butyl hydroperoxide (t-BHP), and sodium azide (NaN3). The cells were then cultured with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) at a final concentration of 1 mg/mL and incubated for 30 min. Dimethyl sulfoxide (200 µL) was then added to each well to dissolve the cells. Absorbance was measured at 570 nm using a Sunrise™ spectrophotometer enzyme-linked immunosorbent assay reader (MRX-TC; Dynex Technologies, Chantilly, VA). Values were corrected for background absorbance by subtracting the appropriate blanks. Data are from five independent assays.
In vitro BL exposure protocol
The light source and cell plates were placed 25 cm apart, as described in our previous study [19]. Luminance was measured with a light meter (LM-81LX; Lutron Electronic Enterprise, Taipei, Taiwan). BLL peaked at 460 nm (60 lux) for the indicated time (0–48 h), depending on the experimental design.
Western blotting
After 0–48-h co-treatment with CTE and BLL exposure, the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and resuspended in a radioimmunoprecipitation assay lysis buffer (1% Nonidet P-40, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM Na3VO4, 1 mM ethylene glycol-bis (β-aminoethylether)-N,N,N′,N′-tetraacetic acid, 1 mM NaF, and protease/phosphatase inhibitor cocktail). The cell lysate was obtained via centrifugation at 12, 000 × g for 35 min, and the supernatant was collected to determine protein concentrations via the Bradford reagent (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein samples were separated by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane. After the membrane was blocked with 5% non-fat milk and incubated in Tris-buffered saline with Tween 20 (TBST) for 1 h, immunoblotting was performed with primary antibodies specific for cleaved caspase-3, phospho-JNK, JNK, phospho-p38, p38, phospho-extracellular signal-related kinase (ERK), ERK, Bcl-2, Bax, Fas-associated protein with death domain (FADD), caspase-8 (GeneTex, Irvine, CA), Fas-ligand (Abcam, Cambridge, UK), and b-actin (Sigma- Aldrich, St. Louis, MO). The blots were then incubated with horse radish peroxidase-conjugated secondary antibodies (1:6000 in TBST; Cayman Chemical, Ann Arbor, MI) for 1 h at room temperature. Using an enhanced chemiluminescence kit (Millipore, Billerica, MA), the intensity of the bands on each blot was analyzed with Image J software after normalizing to b-actin.
Immunofluorescence
RPE cells were fixed with 4% paraformaldehyde in PBS for 15 min. Triton X-100 in PBS (0.2%) was then added to the cells for 15 min, followed by 5% fetal bovine serum in PBS for 30 min; the cells were then incubated with an anti-cleaved caspase-3 antibody at a 1:250 ratio for 24 h (Cell Signaling Technology, Danvers, MA). A goat anti-rabbit IgG secondary antibody (Dylight 594) was used at a ratio of 1:200 (Vector Laboratories, Burlingame, CA). Cell nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI; AAT Bioquest, Sunnyvale, CA). Changes in fluorescence were observed with a laser CS SP5 confocal spectral microscope imaging system (Leica, Teban Gardens, Singapore).
TUNEL staining
DNA fragments, which can be detected by a TUNEL assay, are a significant hallmark of apoptosis. We used an In Situ Cell Death Detection Kit (Cat. No. 11-684-817-910; Roche) to measure apoptotic programmed RPE cell death as per the manufacturer’s protocol. Briefly, we fixed RPE cells with 4% paraformaldehyde and labeled the DNA strand breaks in an enzymatic reaction to detect TUNEL-positive cells, which presented with a green-fluorescent stain. The stained cells were then scanned and analyzed with a Tissue FAXS-plus Imaging System (TissueGnostics, Vienna, Austria). Ten thousand events were counted in the gated RPE region, and we analyzed the images for the number of TUNEL-positive cells with TissueQuest/HistoQuest software (TissueGnostics).
Animal experiments
Male BN rats (300–350 g body weight) were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and kept for 2–3 months. The rats were maintained in a 12-h/12-h light/dark cycle at 26 ± 1°C, 35–47% relative humidity ratio, and ad libitum access to water and food. Untreated rats were maintained in the dark to serve as controls. The study protocols were approved by the Institutional Animal Care Use Committee of Taipei Medical University (approval number: LAC-2016-0442). All experimental procedures involving the use of animals complied with the Association for Research in Vision and Ophthalmology statements for the use of animals in ophthalmic and vision experimental research.
BLL-induced rat model of retinal degeneration
We used a periodic low-luminance long-term BLL exposure-induced model of retinal degeneration, as described in our previous study [19]. Briefly, BN rats were divided into three groups: a control group, which was kept under dark conditions; a BLL-exposed group, which was exposed to periodic BLL in the dark without pupil dilation (460 nm, 150 lux) for 3 h per day for 60 days; and a CTE-treated plus BLL-exposed group, which was pre-treated with CTE (100 mg/kg body weight) for 14 days and then co-treated with periodic BLL exposure for 3 h per day for 60 days. All rats were returned to the animal colonies and the normal light/dark cycle (250 lux, 12 h/12 h) was resumed at the end of the experimental period.
Statistical analysis
Significant differences were calculated using a one-way analysis of variance followed by Tukey’s post hoc test and multiple comparisons test. Statistical significance was determined by p-values of <0.05.
Cistanche tubulosa products
Results
CTE protects RPE cells from oxidative stress damage
To evaluate the cytotoxicity of CTE via the MTT assay, several concentrations of CTE (0–500 μg/mL) were applied to RPE cells. CTE treatment had no obvious toxic effects on RPE cell viability at any of the concentrations tested (Fig. 1A). Therefore, 50 and 100 μg/mL CTE were used in the subsequent experiments. To investigate the anti-apoptotic effects of CTE, different oxidative inducers (H2O2, t-BHP, and NaN3) were co-administered with CTE to RPE cells for 24 h. H2O2, NaN3, and t-BHP decreased cell viability and damaged RPE cells in a dose-dependent manner (slashed bars, Fig. 1B, 1C, and 1D); cell viability was significantly lower in cells treated with 0.03 mM H2O2, 0.3 mM NaN3, and 0.3 mM t-BHP than in the control group.
However, co-treatment with 50 and 100 μg/mL CTE attenuated the H2O2-, NaN3-, and t-BHP- induced RPE cell damage and reversed the decrease in cell viability (black bars, Fig. 1B, 1C, and 1D). The above data suggest that CTE rescues RPE cells from oxidative stress damage.
CTE inhibits BLL-induced RPE cell death
In our previous study, we found that BLL is cytotoxic to RPE cells, which was attributable to changes in the expression of Bcl-2 and Bax. In the present study, we further examined the protective effects of CTE on BLL-induced RPE cell damage. We placed two blue electric LED plates in a cell culture incubator. The distance between the light source and cell plates was 25 cm, and BLL intensity was 60 lux, as measured by a light meter (Fig. 2A). An MTT assay demonstrated that BLL exposure for different periods of time (6–48 h) caused RPE cell death in a time-dependent manner (Fig. 2B). The BLL-induced reductions in cell viability after 24 and 48 h were significantly attenuated when the cells were co-treated with CTE (100 μg/mL) (Fig. 2C). These data suggest that CTE protects RPE cells from BLL-induced phototoxicity.
CTE inhibits BLL-induced apoptosis in RPE cells by regulating the Bcl-2/Bax and Fas/FasL pathways
On the basis of the potential protective effects of CTE in RPE cells, we further investigated the possible involvement of CTE in this process. Two apoptotic signaling pathways were examined, i.e., the Bax/Bcl-2 and Fas/Fas ligand (FasL) pathways, and related proteins (e.g., pro-caspase-8 and pro-caspase-3; Fig. 3A and 3B). BILL exposure (6–48 h) was found to upregulate the protein expression of Bax/Bcl-2, FasL, and FADD in a time-dependent manner (Fig. 3A). Moreover, BLL downregulated the protein expression of procaspase-3 and procaspase-8, resulting in more cleavage forms of caspase-3 and caspase-8 after BLL exposure, which is consistent with our previous study [19]. To evaluate the protective effects of CTE further, different concentrations of CTE were applied to BLL-exposed RPE cells (Fig. 3B). After 48-h BLL exposure, CTE (50 µg/mL) decreased the Bax/Bcl-2 ratio. Further, CTE (100 µg/mL) decreased the protein expression of FasL and FADD, but significantly increased the levels of procaspase-8 and procaspase-3 (Fig. 3C and 3D). Studies have shown that caspase-3 activation is a marker of apoptosis in RPE cells [20]. To confirm the protective effects of CTE further, we performed immunofluorescent staining of cleaved caspase-3. BILL exposure for 48 h was found to induce caspase-3 cleavage and translocation into the nucleus, resulting in RPE cell apoptosis (yellow arrows, Fig. 4A). However, when BLL-exposed cells were co-treated with CTE (100 µg/mL), the BLL-induced expression of cleaved caspase-3 was inhibited. To understand the anti-apoptotic effects of CTE better, we used TUNEL staining to detect DNA fragments in RPE cells. After 48-h BLL exposure, TUNEL-positive cells increased from 1.78 ± 0.36% to 23.82 ± 0.33%; this effect was attenuated by co-treatment with CTE (23.82 ± 0.33% to 2.22 ± 0.11%; Fig. 4B, 4C, and 4D). These results indicate that CTE treatment strongly protects RPE cells from BLL-induced apoptosis by mediating the Bax/Bcl-2 and FasL/FADD pathways.
CTE inhibits BLL-induced phosphorylation of stress response proteins in RPE cells
Previous studies have demonstrated that MAPK pathways, such as ERK, JNK, and p38 MAP, are activated in RPE cells and in the retina after exposure to ultraviolet light or BL [21, 22]. Hence, to investigate the regulatory effects of CTE on the MAPK stress response pathway after BLL exposure, 100 µg/mL CTE was applied to RPE cells under different BLL exposure times (Fig. 5A). Western blot analysis indicated that phosphorylated ERK, JNK, and p38 were activated upon BLL exposure, but significantly inhibited upon co-treatment with CTE in RPE cells (Fig. 5B). The above results indicate that CTE inhibits BLL-induced RPE cell damage, which might be associated with the inhibition of MAPK in RPE cells.
CTE rescues BLL-induced retinal damage after long-term periodic exposure in a rat model
To evaluate the protective effects of CTE, we exposed BN rats to long-term BLL exposure for 60 days and evaluated the effects of CTE. As shown in the schematic presented in Fig. 6A, BN rats were divided into three groups: control group, BLL-exposed group, and CTE plus BLL group (Fig. 6A). In the CTE plus BLL group, BN rats were continuously supplemented with CTE (100 mg/kg body weight) by oral gavage once daily for 14 days before BLL exposure. On day 0, BN rats were co-treated with CTE (100 mg/kg body weight) and exposed to BLL for 3 h per day for 60 days. After 60 days of BLL exposure, the animals were sacrificed for western blot analysis and immunohistochemistry staining. In the CTE plus BLL group, the expression of the procaspase-3 protein in rat eye homogenates was higher after CTE treatment than in the BLL group (Fig. 6B and 6C). These data illustrate that CTE prevents long-term low luminance BLL-induced retinal phototoxicity and apoptosis by inhibiting the activation of cleaved caspase-3. Immunohistochemistry staining provided more evidence regarding the protective effects of CTE on the structure and physiological pathways of the retina (Fig. 7). Further, after BLL exposure we found that the inner neuron layer was diminished and observed morphological deformations at the outer neuron layer (ONL), including the inner segment/outer segment in both the central and peripheral retina, which is consistent with our previous study (Fig. 7B). Importantly, CTE prevented RPE cell deformation, neuronal cell damage, and overall thinning of the retina in the CTE plus BLL group (Fig. 7C). Further, we did not observe TUNEL-positive cells in the central and peripheral retina of normal rats (Fig. 8Ab and 8Bb). Conversely, after BLL exposure for 60 days, TUNEL-positive cells were detected frequently in the ONL of the central and peripheral retina (Fig. 8Ae and 8Be). DAPI staining of the ONL of the central retina was markedly decreased in the BLL group (Fig. 8Ad). However, the presence of TUNEL-positive cells was attenuated and DAPI staining of the central and peripheral retina ONL was increased in the CTE plus BLL group (Fig. 8Ai and 8Bi).
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