Crude Saponins of Panax notoginseng Have Neuroprotective

Article pubs.acs.org/JAFC

Crude Saponins of Panax notoginseng Have Neuroprotective Effects To Inhibit Palmitate-Triggered Endoplasmic Reticulum Stress-Associated Apoptosis and Loss of Postsynaptic Proteins in Staurosporine Differentiated RGC‑5 Retinal Ganglion Cells Dan-dan Wang,†,∥ Hua-zhang Zhu,†,∥ Shi-wei Li,† Jia-ming Yang,† Yang Xiao,† Qiang-rong Kang,‡ Chen-yang Li,‡ Yun-shi Zhao,‡ Yong Zeng,§ Yan Li,§ Jian Zhang,‡ Zhen-dan He,*,‡ and Ying Ying*,† †

Shenzhen University Diabetes Center and ‡Department of Pharmacy, School of Medicine, Shenzhen University, Shenzhen 518060, China § The First Affiliated Hospital of Kunming Medical University, Kunming 650000, China S Supporting Information *

ABSTRACT: Increased apoptosis of retinal ganglion cells (RGCs) contributes to the gradual loss of retinal neurons at the early phase of diabetic retinopathy (DR). There is an urgent need to search for drugs with neuroprotective effects against apoptosis of RGCs for the early treatment of DR. This study aimed to investigate the neuroprotective effects of saponins extracted from Panax notoginseng, a traditional Chinese medicine, on apoptosis of RGCs stimulated by palmitate, a metabolic factor for the development of diabetes and its complications, and to explore the potential molecular mechanism. We showed that crude saponins of P. notoginseng (CSPN) inhibited the increased apoptosis and loss of postsynaptic protein PSD-95 by palmitate in staurosporine-differentiated RGC-5 cells. Moreover, CSPN suppressed palmitate-induced reactive oxygen species generation and endoplasmic reticulum stress-associated eIF2α/ATF4/CHOP and caspase 12 pathways. Thus, our findings address the potential therapeutic significance of CSPN for the early stage of DR. KEYWORDS: CSPN, palmitate, differentiated RGC-5 cells, apoptosis, PSD-95, ER stress, ROS

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INTRODUCTION Overnutrition due to the increased consumption of a high-fat and/or high-carbohydrate diet, and sedentary life as well, is often associated with obesity and type 2 diabetes mellitus (T2DM). A high-fat diet usually results in excessive plasma saturated fatty acids such as palmitate, which can lead to islet β-cell failure, insulin resistance, and subsequently T2DM. T2DM will cause, in turn, an increase in complications, including diabetic retinopathy (DR), which is one of the leading causes of preventable legal blindness worldwide.2 The current therapy strategies for DR are focused on modulating the vascular changes occurring in the advanced stages of DR using laser photocoagulation. However, laser therapy is not sufficient to prevent the progression to blindness and is often associated with considerable side effects.3 Hence, new pharmacological therapies for the early stages of DR are highly demanded. Recently, increasing lines of evidence have suggested that retinal neurodegeneration, including increased neuronal apoptosis, is an early event in the pathogenesis of DR, which could explain some of the functional deficits in vision that occur soon after the onset of diabetes.4,5 Accordingly, new therapeutic interventions based on neuroprotection have been proposed.6,7 Although there is still no consensus on the best pharmacological target for retinal neurodegeneration, apoptosis of retinal ganglion cells (RGCs) has been clearly shown to be involved in the gradual but irreversible loss of ganglion cell bodies and reduction in thickness of the inner retina in humans with diabetes and streptozotocin-induced diabetic rats.8,9 In light of © XXXX American Chemical Society

this possibility, there is an incentive to search for novel agents that can confer neuroprotection against apoptosis of RGCs as options for the early prevention and treatment of DR. Mechanisms behind the apoptosis of RGCs remain largely unknown. Several possibilities, including hyperglycemia, inflammation, oxidative stress, and advanced glycation end products, may contribute to the increased RGC apoptosis in the early stage of DR.10 However, the effect of excessive palmitate levels on apoptosis of RGCs has not been elucidated thus far. Excessive palmitate can induce apoptosis of pancreatic β-cells11 and liver cells12 through various mechanisms including endoplasmic reticulum (ER) stress. A number of insults, such as ischemic and saturated fatty acid,13,14 have been shown to induce protein misfolding in the ER and cause ER stress. The glucose-regulated protein 78 (GRP-78/BiP) is a central regulator of ER function, bound to the luminal side of the ER to the three ER transmembrane sensors: the protein kinase RNA-like endoplasmic reticulum kinase (PERK), the activating transcription factor 6 (ATF6), and the inositol requiring enzyme 1α (IRE-1α). Upon ER stress, BiP is released, allowing aggregation and activation of the transmembrane signaling proteins and induction of the unfolded protein response (UPR) to enhance the degradation of misfolded proteins.15 Activation Received: December 11, 2015 Revised: February 1, 2016 Accepted: February 2, 2016

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DOI: 10.1021/acs.jafc.5b05864 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. HPLC chemical profile of saponins present in CSPN. Dulbecco’s modified Eagle’s minimum essential medium (DMEM) was purchasead from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was from Hyclone (Logan, UT, USA). Streptomycin, penicillin, palmitate, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Staurosporine (STSN) was purchased from Cell Signaling Technology (Boston, MA, USA). A cell counting kit-8 (CCK-8) assay kit was purchased from Dojindo Bio. (Tokyo, Japan). A caspase 3 activity assay kit was obtained from Calbiochem (San Diego, CA, USA). Primary antibodies antiphosphorylated eIF2α, -ATF4, -CHOP, -Bcl-2, -caspase12, and -GAPDH were obtained from Cell Signaling Technology. Primary antibodies anticleaved-caspase3, -BiP, and -Map2 were purchased from Abclonal (Boston, MA, USA), and anti-Brn3a was from Abcam (Boston, MA, USA). Horseradish-peroxidase-conjugated secondary antibodies and Alexa-Fluor-488 or -546 labeled secondary antibodies were obtained from Cell Signaling Technology. All other chemicals were obtained from Sigma-Aldrich. CSPN Preparation. The dried roots of P. notoginseng (8 kg) were smashed and extracted with hot methyl alcohol (MeOH) (32 L × 2) twice under reflux, and then the extracts were concentrated in a rotary evaporator. The MeOH extracts (2.8 kg) were subjected to column chromatography on the highly porous polymer Diaion (HP-20, 30 kg), column size ⌀ 120 mm × lengh 1500 mm, eluting with H2O, H2O/ MeOH (1:1), and MeOH. The elution with 50% H2O/MeOH (26 L) was concentrated in vacuo to obtain the CSPN residues (960 g). Qualitative and Quantitative Analysis of CSPN. The CSPN was analyzed with an Agilent 1260 Infinity HPLC system coupled with a DAD detector, equipped with a quaternary pump delivery system. The separation of CSPN was performed with an Ultimate XB-C18 analytical column (5 μm, 250 × 4.6 mm i.d.), maintained in a thermostat at 25 °C, and the absorbance was measured at 203 nm. The mobile phase was water (solvent A) and acetonitrile (CH3CN, solvent B). The injection volume was 10 μL, and the gradient (A:B = VA:VB) of the mobile phases was as follows: initially, A:B = 81:19, 0−35 min; A:B = 81:19 to 71:29, 35−55 min; A:B = 71:29, 55−70 min; A:B = 71:29 to 60:40, 70−100 min. Total HPLC run time was 100 min, and the flow rate was 1 mL/min. CSPN (20 mg/mL) in methanol and all of the pure compounds (1 mg/mL) in methanol were injected into the C-18 column, respectively, for quantitative analysis. All chromatographic data were acquired and analyzed by using Agilent ChemStation software. A typical HPLC chromatogram of CSPN is shown in Figure 1. The content of four major saponins in CSPN was determined by HPLC using the respective purified ginsenosides Rg1, Re, Rh1, and Rd as standards. The concentrations of these compounds were calculated according to the regression parameters derived from the standard curves. RGC-5 Maintenance and Differentiation. A mouse retinal ganglion cell line RGC-5 was purchased from Yingrun Biotechnology Inc. (NC022, Changsha, China) and maintained as an adherent cell line in DMEM supplemented with glucose (1 g/L), 10% FBS, L-glutamine (4 mM), and a penicillin (100 U/mL)/streptomycin (100 μg/mL) solution in uncoated T-75 tissue culture flasks. RGC-5 cells were induced to differentiate into highly branched, neuron-like cells using a modified protocol developed by Frassetto et al.28 Briefly, RGC-5 cells were allowed to adhere at 37 °C and subsequently treated with the 100 nM staurosporine (STSN, predissolved in DMSO) for 12 h. Following differentiation, the medium was changed and RGC-5

of PERK phosphorylates the eukaryotic initiation elongation factor 2α (eIF2α), reducing the protein load on the ER but facilitating translation of the activating transcription factor 4 (ATF4).16 Under persistent and/or severe ER stress, these proper responses fail to completely alleviate ER stress, and cell apoptosis is triggered. ATF4 promotes activation of the CCAAT-enhancer-binding protein (C/EBP) homologous protein (CHOP),17 a pro-apoptotic factor. In parallel, activation of ER-associated caspase 12 has been implicated under such harsh conditions.18 Emerging evidence suggests that diabetes can induce ER stress and thereby affect the complications of diabetes including DR.19 Recent studies also indicate that ER stress-induced apoptosis is involved in the death of neurons in the retina under different physiological conditions.20,21 Nonetheless, the role of ER stress in the context of palmitate-stressed RGCs has not been determined thus far. Panax notoginseng (Burk.) F. H. Chen, which belongs to the family Araliaceae, is an ancient and well-known traditional Chinese medicine that has been used to boost health, reduce serum lipids, protect hepatocytes, and prolong life in Asia for more than 3500 years. The roots of P. notoginseng are listed in the Chinese pharmacopoeia; these roots contain saponins classified in three categories: protopanaxadiol, protopanaxatriol, and oleanolic types.22 Ginsenosides Rg1, Rh1, Rd, and Re are four major components in the crude saponin fraction of P. notoginseng (CSPN). CSPN has been suggested to exert hepatic- and cerebrovascular-protective effects through biological mechanisms such as antioxidant and anti-apoptosis.23,24 Recently, CSPN has been reported as a promising plant with antidiabetic potential.25 In addition, accumulating evidence has shown that CSPN has remarkable neuroprotective effects on neurons against damages induced by oxidative stress, lipopolysaccharide, etc.26,27 However, the role, if any, of CSPN in protecting RGCs from apoptosis has not been examined. In view of these considerations, the present study was designed to investigate first whether chronic exposure of palmitate has neurotoxic effects on RGCs and whether CSPN has protective effects against palmitate-induced neurotoxicity in RGCs. In addition, we explored whether ER stress provides a mechanistic link in palmitate-induced apoptosis of RGCs and whether CSPN prevents palmitate-induced apoptosis via inhibiting ER stress-associated signaling pathway.

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MATERIALS AND METHODS

Materials. The roots of cultivated P. notoginseng were collected in Wenshan of Yunnan province, China, and authenticated by Prof. Hong Xu, Hong Kong University of Science and Technology. A voucher specimen has been deposited by the School of Medicine, Shenzhen University, Shenzhen, China. The standards of ginsenosides Rg1, Re, Rh1, and Rd were isolated from the roots of P. notoginseng, and their structures were identified by our research group, with purities of >98%. B


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Figure 2. Structures of four major peaks detected in the HPLC profile of CSPN. cells were grown for an additional 12−60 h and analyzed for the expression of neuronal markers by immunofluorescence or used immediately for subsequent experiments. Experimental Design. After treatment with STSN for 12 h, RGC-5 cells were incubated with palmitate (400 μM) preconjugated to BSA in 0.5% FBS−DMEM, in the absence or presence of CSPN. The stock solution of 5 mM palmitate bound to 10% BSA was prepared using the methods described before.29 The final concentration of palmitate in the stock solutions was determined before and after sterile filtration with a commercially available kit (Wako Chemicals, Neuss, Germany). For control incubations, 10% BSA was also prepared. Cell Viability Assay. Cell viability was measured using a watersoluble tetrazolium-8 based cell counting kit-8 (CCK-8) assay kit. The absorbance was measured at 450 nm using an automated microplate reader. Hoechst 33342 Staining. Cells were loaded with 1 μM Hoechst 33342 (predissolved in H2O) for 10 min at 37 °C. The fluorescence was observed with an Olympus FV1000 confocal laser scanning microscope. Nuclei with Hoechst staining nuclear condensation, shrinkage, and even collapse were considered as apoptotic nuclei. Apoptotic cells were counted in 10 different microscopic fields of at least three independent experiments. Caspase 3 Activity Analysis. Caspase 3 activity was measured using a colorimetric assay kit following the manufacturer’s instructions. The proteolytic reaction was performed in protease assay buffer

containing 2 mM acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVDpNA). Cleavage of the Ac-DEVD-pNA fluorophore was determined in a microplate reader (Eppendorf, Germany). Caspase 3 activity was calculated as units of fluorescence (mg of protein × h)−1. Indirect Immunofluorescence. RGC-5 cells cultured on glass coverslips were fixed with 4% paraformaldehyde and permeabilized in 0.1% Triton X-100. After blocking unspecific binding by incubation for 30 min with 1% BSA, cells were incubated overnight at 4 °C with the respective anti-Map2, -Brn3a, -BiP, or -CHOP primary antibody (1:100 diluted in 1% normal donkey serum in PBS). Cells were then incubated at 37 °C for 1 h with an Alexa-Fluor-488 or -546 labeled secondary antibody (1:100 in PBS) and counterstained with Hoechst 33342. Images were obtained with an Olympus FV1000 confocal microscope. Western Blot Analysis. Forty micrograms of cellular protein was resolved by 10% or 12% SDS−polyacrylamide gel electrophoresis and then electroblotted onto polyvinylidene fluoride membranes. The membrane was immunoblotted with the respective anti-phosphorylated eIF2α, -BiP, -ATF4, -CHOP, -Bcl-2, -cleaved-caspase 3, -caspase 12, or -GAPDH primary antibody (1:1000) at 4 °C overnight, followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 1 h. Immunoreactive bands were revealed by enhanced chemiluminescence and visualized by the Kodak Image Station 4000MM PRO. Band intensities were quantified by scanning C


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Journal of Agricultural and Food Chemistry densitometry (Gel-Doc2000, Bio-Rad) and analyzed with Quantity One (Bio-Rad). Measurement of Intracellular ROS Production. To evaluate intracellular reactive oxygen species (ROS) production, differentiated RGC-5 cells seeded on a glass coverslip and treated under different conditions were incubated with 1 mL of serum-free DMEM containing 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) for 1 h at 37 °C under 5% CO2. Cells were subsequently fixed and observed under an Olympus FV1000 confocal microscope. Fluorescence intensity was quantified per cell in at least 200 cells, using Image-Pro Plus software. Statistical Analysis. All data were expressed as mean ± standard error of the mean (±SEM). Statistical significance was analyzed with an unpaired Student’s t test. Data were considered significant when P < 0.05.

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RESULTS Analysis of Constituents and the Content of CSPN. In the present study, residues of the CSPN were extracted from the roots of P. notoginseng as described under Materials and Methods. The chemical profile of CSPN was analyzed by HPLC (Figure 1). The identities of four major peaks in the HPLC profile of CSPN were determined by retention times of the corresponding standard. The structures of four major constituents shown in Figure 2 were illustrated by spectrometric methods and by comparion with data from previous studies.30,31 The contents of compounds 1−4 are shown in Table 1. Ginsenoside Rg1 (1) and ginsenoside Rh1 (3) were

Figure 3. Differentiation of RGC-5 cells with staurosporine. RGC-5 cells (5 × 104/well) were plated onto the coverslips in 6-well plates. Cells were allowed to adhere for 1 h at 37 °C and subsequently treated with STSN (100 nM) for 12 h. Following differentiation, the medium was changed, and cells were grown for an additional 12−60 h. (A) Representative bright-field images of RGC-5 cells at 12, 24, 48, and 72 h following adherence. (B) Expression of neuronal markers examined by indirect immunofluorescence (green, Map2; red, Brn3a; blue, Hoechst 33342) at 12, 24, 48, and 72 h following adherence. Mock, carrier solution treated RGC-5 cell. STSN trt, differentiation of RGC-5 cells with staurosporine for 12 h. Scale bar = 50 μm.

Table 1. Contents of Four Major Saponins Present in CSPN Isolated from the Roots of Panax notoginsenga compound 1 2 3 4 a

content (mg/100 mg in CSPN) 23.77 1.75 16.85 5.01

± ± ± ±

0.15 0.15 0.25 0.22

Average of three measured data by HPLC.

RGC-5 cells, whereas less abundance of Map2 was detected in untreated RGC-5 cells. In addition, the expression of the RGC specific marker Brn3a was readily detected in differentiated RGC-5 cells, whereas lack of Brn3a staining was found in untreated RGC-5 cells (Figure 3B). Thus, these results revealed that STSN-differentiated RGC-5 cells developed a retinal neuron-like phenotype and expressed neuronal markers consistent with RGCs in vivo, which was used as an in vitro model for elucidating the potential neuroprotective effects of CSPN in the subsequent experiments. CSPN Attenuated Palmitate-Induced Cell Death of Differentiated RGC-5 Cells. Initially, we examined whether palmitate or CSPN affects the viability of STSN-differentiated RGC-5 cells using a CCK-8 assay (Figure 4A,B). Stimulation of differentiated RGC-5 cells with BSA-conjugated palmitate decreased cell viability in a dose-dependent (0−400 μM) and time-dependent (0−36 h) manner. We found that palmitate stimulation up to 400 μM for 36 h demonstrated a maximum toxic effect on differentiated RGC-5 cells as compared with control (Figure 4A). Therefore, incubation of palmitate at 400 μM for 36 h was adopted for subsequent determination of palmitate-induced RGC damage in the present study. We then evaluated the effect of CSPN alone on the cell viability by exposing differentiated RGC-5 cells to various concentrations (0−1200 μg/mL) of CSPN for 36 h. Treatment of CSPN up to 800 μg/mL did not appear to have a negative effect on differentiated RGC-5 cell viability, whereas there was an

the two major constituents. The amount of four major compounds in Table1 accounted for ∼47.11% of CSPN according to the analysis of HPLC. The protective effects of CSPN on palmitate-stressed RGCs were investigated in the subsequent experiments. Staurosporine-Induced Differentiation of RGC-5 Cells. Establishing an in vitro model of RGCs is vital for studying the potential neuroprotective effects of CSPN. However, isolation of primary RGCs results in a limited number of cells that will survive for only a few days. Currently, RGC-5 represents the only available transformed cell line that retains some characteristics of primary RGCs.32 In our experimental setup, RGC-5 cells were induced to differentiate into a neuronal phenotype using a modified protocol.28 As shown in Figure 3A, naı̈ve, untreated RGC-5 cells showed flat, elongated, and polygonal morphologies with few extensions emanating from the cell body 12−72 h following adhering. In contrast, RGC-5 cells treated with 100 nM STSN for 12 h changed their phenotype to neuron-like and maintained these highly branched morphologies for up to 72 h in culture. We further characterized the RGC-5 cells by determining the expression of neuron-specific microtubule-associated protein 2 (Map2) and RGC-specific transcription factor Brn3a that is expressed by RGC in vivo. Fluorescence images clearly showed that Map2 was abundantly expressed in the cell bodies and neurites in STSN-differentiated D


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palmitate induces apoptosis of differentiated RGC-5 cells through Hoechst 33342 staining. Compared with control, about 50% of cells treated with palmitate showed chromatin condensation and/or fragmentation (Figure 5A). Quantification of apoptotic nuclei revealed that palmitate significantly increased the number (∼4.7-fold, P < 0.01) of apoptotic cells (Figure 5B). We also evaluated a critical mediator of the execution phase of cell apoptosis, caspase 3. Palmitate induced a markedly increased activation of caspase 3 activity (∼3.2-fold, P < 0.01) as compared to control (Figure 5C). Moreover, protein levels of cleaved caspase 3 were significantly increased in palmitate-stressed differentiated RGC-5 cells (Figure 5D). We next investigated whether CSPN prevents palmitateinduced apoptotic cell death. Treatment with CSPN dosedependently (100−400 μg/mL) reduced chromatin condensation and/or fragmentation (Figure 5A,B), activation of caspase 3 activity (Figure 5C), and cleavage of caspase 3 (Figure 5D) in palmitate-stimulated RGC-5 cells. Again, the highest dose of CSPN (800 μg/mL) did not exhibit improved anti-apoptotic effect against palmitate, as compared to the lower doses (200 and 400 μg/mL). Strikingly, the elevated apoptosis in palmitate-stressed RGC-5 cells was almost reversed by CSPN at a concentration of 400 μg/mL, as compared to control (Figure 5B−D). Thus, 400 μg/mL CSPN was used for further experiments. Collectively, these findings validated that CSPN can prevent palmitate-induced apoptosis of differentiated RGC-5 cells. CSPN Restored Palmitate-Caused Loss of Postsynaptic Protein PSD-95 in Differentiated RGC-5 Cells. PSD-95 is an important postsynaptic scaffolding protein playing a critical role in protein assembly, synaptic development, and neural plasticity.33 We then examined the effect of CSPN on the expression of PSD-95 in palmitate-stressed RGC-5 cells. Immunofluorescence microscopy analysis showed that palmitate induced an extensive loss of PSD-95 expression, whereas CSPN (400 μg/mL) greatly rescued palmitate-reduced PSD-95 expression in RGC-5 cells (Figure 6A). Western blot analysis further confirmed that the levels of PSD-95 were significantly decreased in palmitate-stimulated RGC-5 cells, as compared to untreated cells. However, CSPN significantly restored the reduced PSD-95 levels by palmitate (Figure 6B), suggesting a role of CSPN in rescuing loss of postsynaptic proteins during chronic palmitate exposure to RGC-5 cells. CSPN Inhibited Palmitate-Triggered ER Stress in Differentiated RGC-5 Cells. Previous studies reported that chronic exposure of palmitate can induce apoptosis through triggering ER stress.11,12 Drugs inhibiting ER stress-induced apoptosis are an important strategy for providing efficient neuroprotective effects. We thus elucidated whether ER stress is involved in palmitate-induced apoptosis and whether CSPN attenuates palmitate-induced ER stress in differentiated RGC-5 cells. Compared to untreated cells, a significant increase in protein expression of the endogenous ER stress marker, BiP, was observed in palmitate-stimulated differentiated RGC-5 cells, whereas treatment with CSPN (400 μg/mL) remarkably suppressed this induction (Figure 7A). Immunofluorescence microscopy analysis also revealed that CSPN markedly prevented palmitate-mediated accumulation of BiP in differentiated RGC-5 cells (Figure 7B). In addition, palmitate significantly increased phosphorylation of eIF2α, whereas CSPN markedly attenuated palmitate-induced eIF2α phosphorylation. Concomitantly, CSPN treatment reversed palmitatetriggered ATF4 protein expression in differentiated RGC-5 cells

Figure 4. CCK-8 assay determined the protective effects of CSPN on palmitate-induced cytotoxicity in differentiated RGC-5 cells. (A) Doseand time-dependent effect of palmitate on differentiated RGC-5 cell viability. Cells were exposed to media containing BSA alone (Ctrl) or different concentrations (200 and 400 μM) of BSA-conjugated palmitate (PA) for 0−36 h, respectively. Values are expressed as percentages of cell viability at Ctrl. Data are means ± SEM of three independent experiments in duplicate. (∗) P < 0.05 and (∗∗) P < 0.01 versus Ctrl; (#) P < 0.05 and (##) P < 0.01 versus 200 μM of PA. (B) Effects of various concentrations of CSPN (100−1200 μg/mL) on differentiated RGC-5 cell viability. Values are expressed as percentages of cell viability at Mock (without CSPN treated). Data are means ± SEM of three independent experiments in duplicate. (∗) P < 0.05 versus Mock. (C) Differentiated RGC-5 cells cotreated with 400 μM PA and increasing amounts of CSPN (0−800 μg/mL) for 36 h. Values are expressed as percentages of cell viability at negative control group (without PA and CSPN treatment). (∗) P < 0.05 and (∗∗) P < 0.01 versus negative control; (#) P < 0.05 and (##) P < 0.01 versus PA alone.

obvious cytotoxic effect of CSPN at 1200 μg/mL (Figure 4B). Thus, we chose the concentrations of CSPN at 100, 200, 400, and 800 μg/mL for further exploring the cytoprotective effects of CSPN against palmitate. Next, we examined whether CSPN protects against palmitateinduced retinal neuronal cell death by co-administering palmitate (400 μM) and increasing amounts of CSPN (100−800 μg/mL) to differentiated RGC-5 cells for 36 h. As shown in Figure 4C, treatment with CSPN significantly and dose-dependently (100−400 μg/mL) attenuated palmitate-induced differentiated RGC-5 cell death. Interestingly, the highest dose of CSPN (800 μg/mL) did not exhibit improved protective effect against palmitate, as compared to the lower dose (400 μg/mL). Our data thus confirmed that CSPN possesses a protective ability against palmitate-caused cell death of differentiated RGC-5 cells. CSPN Prevented Palmitate-Mediated Apoptosis of Differentiated RGC-5 Cells. We further determined whether E


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Figure 5. CSPN prevented palmitate-induced apoptosis of differentiated RGC-5 cells. Differentiated RGC-5 cells were treated with 400 μM PA, in the absence or presence of increasing amounts of CSPN (100−800 μg/mL) for 36 h. (A) Representative images of Hoechst-33342 labeled nuclei. Apoptotic nuclei showing nuclear condensation, shrinkage, or even collapse are indicated by white arrows. Scale bar = 50 μm. (B) Quantification of apoptotic nuclei by Image-Pro Plus software. Values are expressed as the percentage of apoptotic to total nuclei. (C) Caspase 3 activity measured using a colorimetirc assay kit and calculated as units of fluorescence (UF/mg protein × h). (D) Western blot analysis for cleaved caspase 3. Intensities of protein expression were quantified, normalized against the level of GAPDH and expressed as fold of protein abundance at negative control (without PA and CSPN treatment). Data are means ± SEM of three independent experiments. (∗) P < 0.05 and (∗∗) P < 0.01 versus negative control; (#) P < 0.05 and (##) P < 0.01 versus PA alone control. PA, palmitate.

(Figure 7A). These findings suggested that chronic exposure of palmitate strongly activated the eIF2α/ATF4-dependent ER stress pathway, which was inhibited by CSPN treatment. Persistent ER stress can trigger apoptosis via activating CHOP, an ATF4 downstream target protein, or ER-localized cysteine protease, caspase 12.17,18 As shown in Figure 7A, palmitate-induced ER stress triggered a significant increase in the protein expression of CHOP by >4-fold compared to control, whereas CSPN markedly inhibited palmitate-induced CHOP expression in RGC-5 cells (Figure 7A). Moreover, immunofluorescence analysis showed that CSPN greatly suppressed palmitate-triggered enhanced expression and nuclear translocation of CHOP in differentiated RGC-5 cells (Figure 7C). CHOP was reported to mediate ER stress-induced apoptosis partly by down-regulating the anti-apoptotic Bcl-2.34 Consistent with previous studies, we found a significant decrease in Bcl-2 expression in palmitate-stimulated RGC-5 cells, which was restored by CSPN treatment (Figure 7A). In parallel, palmitate also induced a weak but significant activation of caspase 12, whereas CSPN considerably attenuated palmitatestimulated cleavage of caspase 12 (Figure 7A), suggesting a possible participation of both CHOP and caspase 12 in the ER stress-induced apoptosis of differentiated RGC-5 cells.

CSPN Attenuated Palmitate-Induced ROS Production in Differentiated RGC-5 Cells. Recently, one proposed mechanism of palmitate-induced ER stress is the generation of ROS.35 To evaluate the effect of CSPN on palmitate-induced ROS, differentiated RGC-5 cells were treated with palmitate for 2−24 h in the presence or absence of CSPN (400 μg/mL). Single-cell changes in ROS content were assessed by the redoxsensitive dye DCFH-DA under immunofluorescence microscopy. Palmitate significantly increased ROS generation from 6 to 24 h compared to untreated cells, whereas CSPN remarkably suppressed this induction over time (Figure 8), suggesting that CSPN could effectively prevent palmitate-induced ER stress at least partly by inhibiting ROS generation.

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DISCUSSION Increasing lines of evidence suggest that neurodegenerative changes occur beyond the microcirculatory abnormalities of the retina at the early stage of DR. Recently, increased apoptosis of RGCs has been found to contribute to the gradual loss of retinal neurons and functional deficits in the vision of humans with diabetes.8,9 Although high levels of plasma saturated fatty acid such as palmitate have been proposed as a potential mechanism for the development of diabetic F


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Figure 6. CSPN prevented palmitate-caused loss of postsynaptic protein PSD-95 in differentiated RGC-5 cells. Differentiated RGC-5 cells were incubated with BSA (Ctrl) or treated with palmitate (PA) in the absence or presence of 400 μg/mL of CSPN for 36 h. (A) Representative images of subcellular expression of PSD-95 as examined by indirect immunofluorescence (green, PSD-95; blue, Hoechst-33342; scale bar = 50 μm). (B) Protein levels of PSD-95 determined by Western blot. Intensities were quantified and normalized against the level of GAPDH and expressed as percentages of protein abundance under Ctrl. Data are means ± SEM of three independent experiments. (∗∗) P < 0.01 versus Ctrl; (##) P < 0.01 versus PA.

apoptosis induced by a saturated fatty acid in vitro. Analysis of components and contents of CSPN by HPLC and spectrometric methods revealed that the crude saponin products contained four major ingredients, ginsenosides Rg1, Rh1, Rd, and Re. As is known, the hydrophobic steroid backbone can interact with the hydrophobic side chains of fatty acids and cholesterol of cell membrane, whereas the hydroxyl (−OH) group of ginsenosides allows interactions with the polar head of the cell membrane phospholipids and the β-OH group of cholesterol. These structure features of ginsenosides thus allow the entrance of CSPN into the cells. Of note, recent studies have demonstrated the significant neuroprotective effects of ginsenosides Rg1, Rd, Re, and Re1 in both in vitro and in vivo models of neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases through several mechanisms, including preventing neurons from apoptosis induced by various insults.36−39 Thus, we speculate that ginsenosides Rg1, Rd, Re,

complications, the effect of excessive palmitate on apoptosis of RGCs remains largely unknown. In this study, we showed that prolonged exposure of palmitate markedly promoted the apoptotic cell death of differentiated RGC-5 cells. Thus, our data suggest that high levels of saturated fatty acid may be another important metabolic mechanism associated with the increased apoptosis of RGCs at the onset of DR. The search for drugs that can protect RGCs from apoptosis is an important step toward the development of effective treatment strategies for DR before overt clinical symptoms develop. CSPN has been shown to protect neurons from various damages.14,15 However, little is known regarding the role of CSPN in palmitate-induced damage in retinal neurons. In this study, the residue of CSPN was found to protect RGC-5 cells from palmitate-induced apoptotic cell death and postsynaptic protein loss. This is the first report, to our knowledge, that saponins of P. notoginseng can protect retinal neurons from G


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Figure 7. CSPN inhibited palmitate-triggered ER stress in differentiated RGC-5 cells. Differentiated RGC-5 cells were incubated with BSA (Ctrl) or treated with palmitate (PA) in the absence or presence of 400 μg/mL of CSPN for 36 h. (A) Western blot analysis for BiP, phosphorylated eIF2α, total eIF2α, ATF4, CHOP, Bcl-2, caspase 12, and GAPDH. Intensities were quantified and normalized against the level of total proteins (eIF2α) or GAPDH. Data are means ± SEM of three independent experiments. (∗) P < 0.05 and (∗∗) P < 0.01 versus Ctrl; (#) P < 0.05 and (##) P < 0.01 versus PA. (B, C) Subcellular distribution of BiP (green, Bip; scale bar = 20 μm) or CHOP (red, CHOP; scale bar = 50 μm) examined by indirect immunofluorescence.

of eIF2α, which selectively triggers the expression of ATF4.16 Phosphorylation of eIF2α was associated with the degeneration of neurons40 and mediated the expression of pro-apoptotic protein CHOP by ATF4, resulting in apoptosis.41 Alternatively, prolonged ER stress can activate the ER localized cysteine protease, caspase 12, that is integrated with the caspase 9 and caspase 3 pathways during apoptosis.18,42 In this study, we found that excessive palmitate significantly increased the expression of BiP, phosphorylated eIF2α, ATF4, CHOP, caspase 12, and cleaved caspase 3 in differentiated RGC-5 cells, which, however, was attenuated by CSPN. Thus, another novel finding of our present study is that CSPN can prevent RGC-5 cells from palmitate-induced apoptosis by reducing palmitate-triggerred ER stress via inhibition of eIF2α/ATF4/CHOP-dependent and caspase 12-mediated pro-apoptotic pathways.

and Re1 may account for the anti-apoptosis effect of CSPN on palmitate-stimulated RGC-5 cells. Nevertheless, other components consisting of CSPN besides these four major ginsenosides may be considered to act additively or synergistically in the present actions of CSPN. Therefore, further study is required to clarify the pharmacological action of CSPN at the component level. Previous studies have shown that the mechanism behind the palmitate-induced apoptosis involves the activation of ER stress.17,18 The CHOP-mediated pathway and the caspase 12-mediated pathway are suggested to participate in ER stressinduced neuronal apoptosis. To our knowledge, upon severe or prolonged ER stress, BiP is released and subsequently initiates a series of downstream signaling pathways such as the PERK pathway. Activation of PERK leads to increased phosphorylation H


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Figure 8. CSPN ameliorated palmitate-induced ROS generation in differentiated RGC-5 cells. Differentiated RGC-5 cells were incubated with BSA (Ctrl) or treated with palmitate (PA) in the absence or presence of 400 μg/mL of CSPN for 2−24 h. (A) Representative images of ROS production in cells incubated with DCFH-DA (green; scale bar = 50 μm) at the indicated time periods. (B) Quantification of intracellular ROS production. Values are expressed as the fold increase from Ctrl in ROS content evaluated by fluorescence intensity. Data are means ± SEM of three independent experiments. (∗) P < 0.05 and (∗∗) P < 0.01 versus Ctrl; (#) P < 0.05 and (##) P < 0.01 versus PA.

inputs are collected by the dendrites, which extend into the inner plexiform layer, where abundant synaptic contacts are located. Therefore, the capability of RGCs in performing their vital function rests on the structural and functional integrity of synaptic contacts. PSD-95 is an important postsynaptic scaffolding protein involved in the assembly and function of the postsynaptic density complex.44 Previous studies showed that synaptic PSD-95 levels were decreased in neurons of neurodegenerative diseases such as Alzheimer’s.45,46 Our data here also revealed that levels of PSD-95 were significantly reduced in palmitate-stimulated RGC-5 cells. Activation of ATF4 by phosphorylation of eIF2α was shown to suppress the cAMP response elements binding protein (CREB) activity, resulting in translational suppression of synapse genes.47 Interestingly, the apoptotic executor caspase 3 was found to be increased in postsynaptic densities and trigger early synaptic dysfunction in models of AD.48,49 Thus, the loss of PSD-95 by palmitate in our study might be a synergistic result from the activation of eIF2α/ATF4 and induction of apoptosis in palmitate-stressed RGC-5 cells. Of note, in agreement with its ability to inhibit ER stress-associated eIF2α/ATF4 pathway and apoptosis, CSPN markedly restored palmitate-reduced PSD-95 levels, suggesting that CSPN may have therapeutic potential for preventing palmitate-induced functional deficits and gradual loss of RGCs in early DR. The remarkably protective effects of CSPN on palmitatestressed RGC-5 cells observed in this study also lead us to consider whether the emulsifying property of saponins would make the palmitate less available to the cells. To investigate this possibility, levels of palmiate in RGC-5 culture media before

CHOP is regarded as a critical effector of ER stress-induced apoptosis and was shown to down-regulate the anti-apoptotic Bcl-2 that inhibits the mitochondria-dependent apoptotic pathway.34 In this study, we observed that palmitate-induced expression of CHOP was accompanied by a decreased expression of Bcl-2. In contrast, CSPN treatment restored the expression of palmitate-reduced Bcl-2. The rescue of Bcl-2 by CSPN could prevent the formation of large pores in the mitochondrial outer membrane and the consequent activation of caspase 3, thus protecting RGC-5 cells from palmitateinduced apoptosis. Data from a recent study reveal that a mechanism of palmitate-induced ER stress is the generation of ROS.35 ROS by itself can induce ER stress, whereas severe and prolonged ER stress, such as in the presence of excessive palmitate, can lead to further ROS accumulation due to CHOP activation. As reported, CHOP can induce the expression of Ero-1α, a thiol oxidase that generates ROS.43 The further accumulation of ROS may potentially amplify the apoptotic response. In accordance, our results showed that palmitate induced a gradual increase in ROS generation from 6 to 24 h of incubation, suggesting that besides being a precipitating insult of ER stress, ROS is also a consequence of persistent ER stress. However, treatment with CSPN significantly prevented palmitate-induced ROS generation from as early as 6 h of palmitate incubation, suggesting that CSPN could effectively prevent palmitate-induced ER stress at least partly by inhibiting ROS generation. RGCs are specialized projection neurons that convey visual information from the retina to the brain. In the RGC, signal I


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Figure 9. Schematic of a proposed model for the neuroprotective effects of CSPN on chronic palmitate-stressed differentiated RGC-5 cells and the potential molecular mechanisms.

and after application of CSPN were determined by the gas chromatography−mass spectrometry (GC-MS) analysis. No significant changes of the palmitate concentration were observed (Supplementary Figure 1 and Table 1). Thus, the neuroprotective effect of CSPN was not due to its interaction with palmitate before entering the cells. On the basis of the present findings, we outlined a hypothetical model that explains our findings (Figure 9). Thus, chronic palmitate exposure caused ROS generation, which subsequently induced ER stress, leading to release of BiP from PERK and activation of the PERK/eIF2α/ATF4 signaling pathway. On the one hand, upregulation of ATF4 induces the expression of pro-apoptotic CHOP. CHOP down-regulated the anti-apoptotic Bcl-2 and thus promoted apoptosis, but also induced further generation of ROS, which may potentially amplify the apoptotic response. On the other hand, up-regulation of ATF4 might suppress the expression of CREB-mediated synaptic protein, leading to loss of PSD-95 in RGC-5 cells. In addition, prolonged ER stress by excessive palmitate also activated ER-associated caspase 12, which might also promote apoptosis of RGC-5 cells. Of note, CSPN could block palmitate-induced ROS generation and inhibit the activation of eIF2α/ATF4/CHOP-dependent and caspase 12-mediated pro-apoptotic pathways. Moreover, inhibition of ATF4 activation by CSPN restored the expression of PSD-95 in differentiated RGC-5 cells. Further studies are required to examine the connection between the neuroprotective effects of CSPN on retinal neurons and ER stressassociated signaling in more complex physiological disease processes in vivo. In summary, our present study demonstrated for the first time that CSPN could prevent palmitate-induced apoptosis and loss of postsynaptic proteins in differentiated RGC-5 cells, at least partially by attenuating palmitate-triggered ER stress via mechanisms involving inhibition of ROS generation and

inactivation of eIF2α/ATF4/CHOP- and caspase 12-mediated signaling pathways. Our findings suggest a potential therapeutic target for ER stress-associated eIF2α/ATF4/CHOP and caspase 12 signaling pathways in palmitate-induced retinal RGC apoptosis. Importantly, our results also address the potential therapeutic significance of CSPN in the prevention and treatment of the early phase of DR.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05864.

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Supplementary methods, Figure 1, and Table 1 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*(Y.Y.) Phone: +86-755-86671931. Fax: +86-755-86671906. E-mail: [email protected]. *(Z.H.) Phone: +86-755-86671916. Fax: +86-755-86671906. E-mail: [email protected]. Author Contributions ∥

D.W. and H.Z. made equal contributions.

Funding

This study was supported by grants to Y.Y. from the National Science Foundation of China (No. 81200602) and the Basic Research Foundation of Shenzhen (JCYJ20140418091413550). Z.H. was supported by grants from the Shenzhen Strategic Emerging Industry Development Project (SFG[2013]180, KQCX20140522111508785, CXZZ20150601110000604, ZDSYS201506031617582). Notes

The authors declare no competing financial interest. J


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ABBREVIATIONS USED ATF4, activating transcription factor 4; ATF6, activating transcription factor 6; Bcl-2, B-cell lymphoma-2; CCK-8, cell counting kit-8; CHOP, CCAAT-enhancer-binding protein (C/EBP) homologous protein; CSPN, crude saponins of Panax notoginseng; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; DR, diabetic retinopathy; eIF2α, eukaryotic initiaion elongation factor 2α; ER, endoplasmic reticulum; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GRP78/BiP, glucose-regulated protein 78; HPLC, high-performance liquid chromatography; IRE-1α, inositol requiring enzyme 1α; Map2, microtubule-associated protein 2; MeOH, methyl alcohol; PA, palmitate; PBS, phosphate buffer saline; PERK, protein kinase RNA-like endoplasmic reticulum kinase; PSD-95, postsynaptic density protein-95; RGCs, retinal ganglion cells; ROS, reactive oxygen species; STSN, staurosporine; T2DM, type 2 diabetes mellitus; UPR, unfolded protein response

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Crude Saponins of Panax notoginseng Have Neuroprotective

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