Ganoderma atrum Polysaccharide Ameliorates Hyperglycemia

Article pubs.acs.org/JAFC

Ganoderma atrum Polysaccharide Ameliorates HyperglycemiaInduced Endothelial Cell Death via a Mitochondria-ROS Pathway Wen-Juan Li,† Shao-Ping Nie,† Yu-Fei Yao,‡ Xiao-Zhen Liu,† Deng-Yin Shao,† De-Ming Gong,†,§ Steve W. Cui,†,∥ Glyn O. Phillips,⊥ Ming He,†,# and Ming-Yong Xie*,† †

State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China China People’s Liberation Army No. 94 Hospital, No. 1028, Jinggangshan Avenue, Nanchang 330000, China § School of Biological Sciences, The University of Auckland, Auckland, Private Bag 92019, New Zealand ∥ Guelph Food Research Center, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9 ⊥ Phillips Hydrocolloids Research Centre, Glyndwr University, Wrexham, LL11 2AW Wales, U.K. # Departments of Pharmaceutical Science, Nanchang University, Nanchang 330006, China

Downloaded by UNIV OF NEBRASKA-LINCOLN on September 15, 2015 | http://pubs.acs.org Publication Date (Web): September 8, 2015 | doi: 10.1021/acs.jafc.5b03462

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ABSTRACT: The aim of the present study was to examine the role of Ganoderma atrum polysaccharide (PSG-1) in reactive oxygen species (ROS) generation and mitochondrial function in hyperglycemia-induced angiopathy. In this work, ROS scavenger, oxidizing agent tert-butylhydroperoxide (tBH), mitochondrial permeability transition pore (mPTP) blockers, and caspase inhibition are used to investigate whether PSG-1 may promote survival of human umbilical vein cells (HUVECs) through preventing the overproduction of ROS and mitochondrial dysfunction. Experimental results show that exposure of HUVECs to 35.5 mmol/L glucose increases the proportion of cells undergoing apoptosis. PSG-1, mPTP blocker, or caspase inhibition can reduce apoptosis and ROS generation. PSG-1 also increases mitochondrial Bcl-2 protein formation and mitochondrial membrane potential (ΔΨm) but inhibits Bax translocation, cytochrome c release, and caspase activation. In summary, vascular protection of PSG-1 can be mediated by a mitochondria-ROS pathway. ROS generation and mPTP induction are critical for high glucosemediated apoptosis. PSG-1 ameliorates endothelial dysfunction by inhibiting oxidative stress and subsequent mitochondrial dysfunction. KEYWORDS: active Ganoderma atrum polysaccharide, apoptosis, endothelial cells, mitochondria, reactive oxygen species

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support and antidiabetic effects.8−11 The structure of PSG-1 was characterized and proved as a heteropolysaccharide, mainly (1,3;1,6)-β-D-glucans (Figure 1A).12 However, the effects of PSG-1 on the overproduction of ROS in endothelial cells subjected to high glucose have not been elucidated. Apoptosis is a very important phenomenon that occurs under a wide range of physiological and pathological conditions as part of the cellular mechanism. There is more supportive evidence13,14described for high glucose-induced endothelial cell apoptosis and mitochondrial dysfunction, such as an increase of the permeability of the mitochondrial membrane. Moreover, mitochondria are primary sites for ROS production and a target for oxidative damage. Increased permeability of the mitochondrial membrane leads to apoptotic cell death and results in the release of cytochome c and activation of the mitochondrial apoptotic pathway.15 Previously, we showed that PSG-1 protected against anoxia/reoxygenation-induced apoptosis by inhibiting the activation of the mitochondrial apoptotic pathway and maintaining the mitochondrial function.9 However, the effect of PSG-1 on the mitochondrial dysfunction in endothelial cells exposed to high glucose has not yet been reported.

INTRODUCTION Angiopathy, a major complication of diabetes mellitus, is one of the leading causes of morbidity and mortality in patients with diabetes. Up to 80% of deaths of patients with diabetes are associated with angiopathy. It determines the quality of life and life expectancy of patients with diabetes, and the management of diabetic angiopathy remains a major therapeutic concern.1,2 A recent study has suggested that oxidative stress is most closely related to the onset and progression of diabetic angiopathy. Oxidative stress results from an imbalance between the generation and elimination of the free radical reactive oxygen species (ROS), which is a byproduct of oxidative phosphorylation.3,4 Excessive ROS production can result in degenerative interaction with cellular components leading to apoptotic and necrotic cell death, which leads to clinically distinct disorders including diabetic angiopathy.5 There is increasing interest in the relationship of oxidative stress with diabetic angiopathy, particularly the identification of bioactive natural products, which could act as antioxidants that might mitigate the deleterious action of ROS on biomolecules. These materials then might have potential health benefits during vascular complications of diabetes.6,7 Ganoderma atrum, a member of the genus Ganoderma, is one of the most famous medicinal/nutritional mushrooms in Asian countries. This fungus has attracted considerable attention because its polysaccharide (Ganoderma atrum polysaccharide: PSG-1) possesses biological functions such as cardiovascular © XXXX American Chemical Society

Received: July 15, 2015 Revised: August 31, 2015 Accepted: August 31, 2015

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DOI: 10.1021/acs.jafc.5b03462 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Effects of PSG-1 on high glucose (HG)-induced LDH release and apoptosis in HUVECs. (A) The structure of PSG-1. PSG-1 was proved as a hyteropolysaccharide comprising a backbone of 1,3-linked and 1,6-linked β-Glcp residues substituted at O-3 and O-6 positions as the branch points. The residues of α-1,4-Galp, α-1,2-Manp, and α-1,4-Manp were also found in the backbone. Side chains were terminated by β-Glcp, with the composition of α-1,4-Galp, α-1,4-GalpA, β-1,3-Glcp, and β-1,6-Glcp. (B) Effects of PSG-1 on LDH activity in HUVECs exposed to HG. (C and D) Effects of PSG-1 on the apoptosis in HUVECs exposed to HG. (C) Determination of apoptosis by AnnexinV and PI double staining. (D) Column bar graph of apoptosis. ##P < 0.01 vs the control group; *P < 0.05, **P < 0.01 vs HG group.

Disruption of mitochondria changes the permeability of the mitochondrial membrane and leads to apoptosis in endothelial cells, which indicates that mitochondria play a critical role in vascular complications of diabetes.16 Attention has been focused recently on the role of the mitochondrial permeability transition pore (mPTP) in the regulation of the permeability of the mitochondrial membrane and as a trigger for apoptosis.17 Opening mPTP increases ROS generation, so mPTP might have an important role in ROS signaling. Hence, mitochondrial targeting of ROS generation or mPTP in mitochondria is emerging as a novel approach in the treatment of diabetic angiopathy.18,19

In the present study, we have used an experimental model in which human umbilical vein cells (HUVECs) are exposed to high concentrations of glucose, which partly mimics what happens in vivo in diabetic patients.20 To elucidate the role of PSG-1 in ROS generation and mitochondrial function during hyperglycemia-induced angiopathy, we investigated whether PSG-1 can promote the survival of HUVECs by preventing the overproduction of ROS and mitochondrial dysfunction using the ROS scavengers, the oxidizing agent tert-butyl hydroperoxide (tBH), mPTP blockers, and caspase inhibition. B


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Figure 2. Effects of PSG-1 on high glucose (HG)-induced ROS production and mitochondrial pathology in HUVECs. (A and B) Flow cytometric analysis of ROS generation. A significant decrease of DCF fluorescence was observed in the PSG-1 pretreatment group. (C) Effect of PSG-1 on mitochondrial function by the MTT assay. (D) Effect of PSG-1 on cytochrome c release and the expression of Bcl-2 and Bax by Western blotting analysis. Expressions of the cytosolic and mitochondrial proteins were determined by Western blotting with anti-α-tubulin, anti-VDAC1, anticytochrome c, anti-Bax, and anti-Bcl-2 antibodies. (E and F) Effect of PSG-1 on the loss of ΔΨm subjected to HG. (E) Flow cytometric analysis of ΔΨm as determined by JC-1staining. FL1-H, green; FL2-H, red. (F) The quantitation of ΔΨm was expressed as the ratio of red fluorescence over green fluorescence. **P < 0.01 vs the HG group.

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Biotechnology (Shanghai, China). Annexin V-FITC apoptosis detection kits were from BD Biosciences (San Jose, SD, USA); antiBcl-2, anti-Bax, anticytochrome c, anti-β-actin, and anti-α-tubulin primary antibodies, as well as the HRP-linked secondary antibody, were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2′,7′Dichlorofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes Inc. (Eugene, OR, USA). Anti-VDAC1 rabbit

MATERIALS AND METHODS 12

Reagents. PSG-1 was prepared as previously described. MTT, cyclosporine A (CsA), and N-acetyl cysteine (NAC) were purchased from Sigma (St. Louis, MO, USA). Cell culture products were obtained from Life Technologies (Paisley, Scotland, USA). The 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) assay kit was purchased from Beyotime Institute of C


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Journal of Agricultural and Food Chemistry polyclonal antibody was purchased from Abcam Biotechnology (Abcam, Cambridge, UK). Mitochondria/cytosol fractionation kit and Z-VAD-FMK were obtained from Biovision (Mountain View, CA, USA). Cells and Cell Culture. HUVECs were purchased from the American Type Culture Collection (ATCC, Catalog No: CRL-1730, US). The cells were seeded in 60 mm Petri dishes and cultured in DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) in a humidified incubator (5% CO2, 37 °C). HUVECs, at 80−90% confluence, were treated with a high concentration of glucose (35.5 mmol/L) for the required time of pretreatment with PSG-1 for 1 h before the high glucose treatment. At the indicated time points, the cells were collected for further evaluation. Determination of Cell and Mitochondrial Viability. Cell and mitochondrial viability were determined by modification of a previously published protocol.21 HUVECs were seeded in 96-cell plates at 1 × 104/well for 24 h. The cells were then incubated with high glucose alone or in the presence of PSG-1 for specified times. Cell viability was determined by measuring lactate dehydrogenase (LDH) released from the cells using LDH assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Mitochondrial function was determined by measuring NADPH dehydrogenase activity using the MTT assay. Analysis of Apoptosis. HUVECs were treated with PSG-1 and incubated for prescribed periods. After incubation, HUVECs were harvested and washed with ice-cold PBS, and stained with Annexin V and PI in buffer. Stained cells were analyzed by flow cytometry with a FACSort cell sorter (Becton Dickinson, San Jose, CA, USA). Determination of ROS Generation. After the incubation period, HUVECs were harvested and washed with ice-cold PBS. Washed cells were further incubated with10 μM DCFH-DA at 37 °C for 20 min in dark. Cells were washed in PBS and analyzed by flow cytometry with a FACSort cell sorter. Measurement of Mitochondrial Membrane Potential (ΔΨm). Flow cytometry with a JC-1 fluorescent probe was used to determine the change of ΔΨm in HUVECs. The HUVECs were harvested and washed with 500 μL of culture medium, and then 500 μL of JC-1staining fluids was added to the cells according to the manufacturer’s instructions. ΔΨm was expressed as a ratio of red fluorescence intensity over green fluorescence intensity. SDS−PAGE and Western Blot Analyses. SDS−PAGE and Western blot analyses of Bcl-2, Bax, or cytochrome c were performed as previously described.8,22 Anti-α-tubulin and anti-VDAC1 antibodies were used as total control, cytosolic loading control, and mitochondrial loading control, respectively. Determination of mPTP Opening. The opening of mPTP was determined using the mPTP fluorescence detection kit (Genmed Scientific Inc., USA). After the indicated treatment, the fluorescence intensity of samples was determined by using a Varioskan Flash Multimode Reader (Thermo Electron Corporation, USA). Assay of Caspase Activities. Caspase activities were measured by a colorimetric assay kit (Biovision, CA, USA). The absorbance at 405 nm of the released ρNA was monitored spectrophotometerically. The protein contents were measured by a BCA kit (Beyotime Company, Nanjing, China). Statistical Analyses. All data are expressed as the mean ± SEM. Statistical significance was tested by one-way analysis of variance. A value of P < 0.05 was considered to be statistically significant.

PSG-1 significantly decreased the amount of LDH released. Such PSG-1 pretreatment caused no significant cytotoxicity in normal cells. To avoid the effect of osmotic pressure on cell injury, mannitol (5.5 mmol/L glucose + 30.0 mmol/L mannitol) was used as a control (Figure 1B). The data showed that mannitol did not protect against high glucose-induced cytotoxicity in HUVECs. These results indicated that PSG-1 was a protective agent against high glucose-induced injury in HUVECs. Effects of PSG-1 on High Glucose-Induced Apoptosis in HUVECs. Apoptosis was determined by Annexin V and PI double staining. Figure 1C and D showed a significant increase of apoptosis in endothelial cells exposed to high glucose. However, when cells had been pretreated with PSG-1 for 1 h before being subjected to high glucose, a decrease in the number of apoptotic cells was observed. These data suggested that PSG-1 could protect endothelial cells against apoptosis induced by high glucose. Effects of PSG-1 on High Glucose-Induced ROS Production in HUVECs. Our recent studies have suggested that overproduction of ROS could be attenuated by PSG-1 both in vivo and in vitro.9,23 To further analyze the effects of PSG-1 on ROS production, HUVECs were incubated with PSG-1 prior to being exposed to high glucose. It was found that PSG-1 significantly reduced 2′,7′-dichlorofluorescein (DCF) fluorescence, suggesting a significant decrease of ROS production (Figure 2A and B). We considered that this showed a link between the attenuation of intracellular oxidative stress by PSG-1 and its protective effect against hyperglycemia toxicity. To find the relationship between the overproduction of ROS and apoptosis, we used NAC, a ROS scavenger, to investigate the role of ROS in apoptosis after HUVECs had been cultured under elevated glucose concentration conditions. Confirming that glucotoxicity is related to oxidative stress resulting from ROS generation, pretreatment of endothelial cells with 15 mmol/L NAC for 1 h, significantly ameliorates high glucose-induced apoptosis (Figure 1C and D) and ROS generation (Figure 2A and B). Effects of PSG-1 on High Glucose-Induced Mitochondrial Dysfunction in HUVECs. Mitochondria are believed to play a crucial role in controlling ROS generation, and its dysfunction is one of the prominent phenomenons of ROSinduced cell damage.24 We further examined the effects of PSG1 on mitochondrial dysfunction induced by high glucose in HUVECs. As shown in Figure 2C, treatment with high glucose for 48 h induced a significant loss of the activity of mitochondrial dehydrogenase, this points to mitochondrial impairment, which could be significantly attenuated by the addition of PSG-1. The maintenance of the ΔΨm is also crucial for mitochondrial functions such as ATP generation and electron transport. Previously, we showed that the bioactivity of PSG-1 is a factor in maintaining ΔΨm in cardiomyocytes exposed to anoxia/reoxygenation.9 In this work, high glucose led to a significant decrease in the red (JC-1 aggregates FL2-H +/FL1-H+)/green (JC-1 monomers FL2-H−/FL2-H+) ratio, suggesting the loss of ΔΨm in endothelial cells (Figure 2E and F). Significantly, the loss of ΔΨm was blocked by PSG-1 pretreatment, indicating that PSG-1 could alleviate hyperglycemia-promoted mitochondrial dysfunction by both increasing the activity of mitochondrial dehydrogenase and maintaining ΔΨm in HUVECs. Effects of PSG-1 on the Mitochondrial Apoptotic Pathway in HUVECs Subjected to High Glucose. Over-

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RESULTS Effects of PSG-1 on High Glucose-Induced Cytotoxicity in HUVECs. Increase of LDH release is widely used as an index of cellular necrosis.21 To investigate the effect of PSG-1 on high glucose-induced cell damage, we treated HUVECs for 48 h with a high concentration of glucose (35.5 mmol/L) alone or after pretreatment with PSG-1 (50 μg/mL) for 1 h.11 HUVECs subjected to high glucose insults induced a significant increase of LDH release. Simultaneous treatment of cells with D


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Figure 3. Effects of PSG-1 on mPTP opening, ROS generation, and apoptosis in HUVECs subjected to high glucose (HG) or BHT. (A) Effects of PSG-1 on mPTP opening in HUVECs subjected to HG. The opening of mPTP was analyzed by calcein fluorescence. The decrease of calcein fluorescence suggested the opening of mPTP. (B) Effects of PSG-1 on mPTP opening in HUVECs subjected to BHT. (C and D) Effects of PSG-1 on ROS generation in HUVECs subjected to BHT. (E and F) Effects of PSG-1 on apoptosis in HUVECs subjected to BHT. ##P < 0.01 vs the control group; *P < 0.05, **P < 0.01 vs the HG group; and P < 0.05 and P < 0.01 vs the BHT group.

after 48 h of treatment with high glucose. However, cytochrome c release from mitochondria into cytosol was significantly reduced when HUVECs were incubated with PSG1 before high glucose treatment (Figure 2D). Bax and Bcl-2, the two main members of the Bcl-2 family, act as an upstream checkpoint of mitochondrial dysfunction associated with cytochrome c release.27 Western blotting

production of ROS particularly affects the mitochondria, which causes the collapse of the ΔΨ), triggering the release of mitochondrial cytochrome c to activate the mitochondrial apoptotic pathway.25,26 The data showed that cytochrome c in control mitochondrial cells was highly expressed, with very low expression in cytosol, but cytochrome c diffused out of the mitochondria and was significantly accumulated in the cytosol E


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Figure 4. Effects of PSG-1 or CsA on ROS generation and apoptosis in HUVECs subjected to high glucose (HG). (A) and (B) Effects of PSG-1 or CsA on ROS generation in HUVECs subjected to HG. (C and D) Effects of PSG-1 or CsA on apoptosis in HUVECs subjected to HG. ##P < 0.01 vs the control group; *P < 0.05, **P < 0.01 vs the HG group.

An oxidizing agent, tBH, which is known to cause mPTP opening in living cells,29 was also used to investigate the opening of mPTP in HUVECs. There was a remarkable increase in apoptosis accompanying the opening of mPTP in HUVECs treated with 500 μmol/L tBH for 45 min. This result confirms that mPTP is an oxidative stress-sensitive channel related to apoptosis (Figure 3B). When HUVECs were subjected to PSG-1 for 1 h, the tBH-induced opening of mPTP decreased significantly in parallel with the attenuation of ROS generation (Figure 3C and D) and apoptosis (Figure 3E and F). These results suggested that PSG-1 may ameliorate oxidative stress-related mPTP opening in HUVECs. Next, we examined whether manipulation of mPTP opening may alter ROS production. Treatment of the reference mPTP inhibitor CsA resulted in the inhibition of ROS generation (Figure 4Aand B) and also alleviated apoptosis (Figure 4C and D) in HUVECs exposed to high glucose. This would suggest that manipulation of mPTP opening could influence ROS production. Similar results were observed with PSG-1, which was also found to inhibit ROS generation and apoptosis. These findings suggested that mPTP opening was a necessary factor for apoptosis and was closely related to ROS generation. Inhibition of mPTP opening may, therefore, be a means of protecting PSG-1 against hyperglycemia toxicity-induced apoptosis in HUVECs.

analysis showed that Bcl-2 expression was significantly reduced from the mitochondria after high glucose treatment (Figure 2D), but after treatment with high glucose for 48 h, an increased amount of Bax was found in the mitochondrial fraction of HUVECs, whereas the protein of cytosolic Bax was decreased with this treatment. In contrast, PSG-1 alleviated the changes of antiapoptotic Bcl-2 and proapoptotic Bax in HUVECs under high glucose conditions. Meanwhile, PSG-1 inhibited the release of cytochrome c into the cytosol and alteration of Bcl-2 paralleled with the increase of ΔΨm and the attenuation of apoptosis. The results indicated that high glucose triggered the mitochondrial apoptotic pathway, which was suppressed by PSG-1 in HUVECs. Effects of PSG-1 on mPTP Opening in HUVECs Subjected to High Glucose. Opening of the mPTP is a mechanism which releases apoptotic factors (such as cytochrome c) and mitochondrial membrane permeabilization as occurs during apoptosis.28 Induction of mPTP opening results in a decrease of calcein fluorescence, and the decrease of fluorescence intensity corresponds to such opening. The results showed that high glucose markedly enhanced the opening of mPTP (Figure 3A). Significantly, PSG-1 inhibited the opening of mPTP, indicating that its effect against apoptosis may be associated with inhibiting the opening of mPTP in HUVECs when exposed to high glucose. F


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Figure 5. Effects of PSG-1 on caspase activity in HUVECs subjected to high glucose (HG). (A and B) Effects of PSG-1 on the activities of caspase-3 and caspase-9. (C and D) Effects of caspase inhibitor “Z-VAD-FMK” or PSG-1 on apoptosis in HUVECs subjected to HG. ##P < 0.01 vs the control group; *P < 0.05, **P < 0.01 vs the HG group.

Effects of PSG-1 on Caspase Activity in Endothelial Cells. Caspase activation is believed to play an important role in the execution of apoptosis.30 We found that the activities of caspase-3 and caspase-9 were significantly increased by the high glucose treatment (Figure 5A and B). PSG-1 was found to attenuate the activation of both caspases produced by high glucose. PSG-1 therefore may protect endothelial cells against apoptosis by attenuation of caspase activation. Meanwhile, high glucose-induced apoptosis (Figure 5C and D) was alleviated by treatment with caspase inhibitor “Z-VAD-FMK” (20 μmol/L). These results suggested that high glucose-stimulated apoptosis was caspase-dependent and that PSG-1 could reduce caspase activation.

hyperglycemia-induced angiopathy, but its role in the apoptotic process has not been elucidated. Our results have demonstrated a rapid increase of the ROS levels is associated with a significant increase of apoptosis. This would indicate that enhancement of ROS production is an essential factor for hyperglycemiainduced apoptosis in endothelial cells. Because enhancement of ROS generation can cause apoptosis in high glucose conditions, we have, therefore, examined whether inhibiting ROS production with the potent antioxidant and free radical scavenger NAC can prevent apoptosis in sustained high glucose systems.32,33 Our results showed that addition of NAC did inhibit apoptosis and added to the evidence that oxidative stress resulting from overproduction of ROS plays a crucial role in hyperglycemia-induced apoptosis. Having demonstrated that PSG-1 did alleviate oxidative stress by the attenuation of ROS generation both in vitro and in vivo,9,10 we have therefore further investigated whether endothelial cells are similarly influenced in the context of hyperglycemia. We found that decreased levels of ROS generation led to a reduction in apoptosis and cell injury, indicating that attenuation of ROS generation could enable PSG-1 to be effective against diabetic angiopathy. The mitochondria generate a steady stream of ROS, which is particularly susceptible to oxidative stress.34 In the present study, we found that high glucose-treated endothelial cells showed marked changes in several parameters of the mitochondrial function. Interestingly, these changes were

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DISCUSSION Diabetic angiopathy has been defined as endothelial dysfunction and is recognized as the main complication of diabetes.31 The present study was designed to investigate the role of a novel Ganoderma atrum polysaccharide, PSG-1, in the cell damage of hyperglycemic endothelial cells. We have shown, for the first time, that PSG-1 inhibits endothelial dysfunction by ameliorating mitochondrial damage and apoptosis in high glucose-stimulated endothelial cells. There is also mounting evidence that hyperglycemia-induced oxidative stress resulting from the overproduction of ROS is a major factor contributing to the development and progression of diabetic angiopathy. Overproduction of ROS is a factor in G


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Figure 6. Schematic model for the cellular mechanisms of PSG-1 on the amelioration of apoptosis induced by high glucose in HUVECs. Mitochondria are the primary source of ROS, which are produced by the mitochondrial electron transport chain (ETC). A low level of ROS was generated by the mitochondrial electron transport chain (ETC), and mPTP remains closed under normal conditions. However, under high glucose conditions, ROS are generated in excess and raised to a level that leads to the opening of mPTP. Moreover, the opening of mPTP could trigger the enhancement of ROS generation. These changes coincide with the alteration of Bcl-2 family and the loss of ΔΨm, resulting in cytochrome c release and caspase-9 activation. Caspase-9 activation in turn triggers the caspase-3 activation, which subsequently executed apoptosis. Accordingly, PSG-1 could ameliorate endothelial cell damage in response to high glucose via preventing the overproduction of ROS and opening of mPTP. Red circle, cytochrome c; IMS, intermembrane space; OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane; ICAD, inhibitor of caspase-activated deoxyribonuclease; CAD, Caspase-activated deoxyribonuclease.

includes both antiapoptotic and proapoptotic proteins, acts as a checkpoint upstream for mitochondrial apoptosis. Antiapoptotic proteins promote cell survival, whereas proapoptotic proteins mediate mitochondria-dependent apoptosis. Bax and Bcl-2, the two main members of this family, influence the permeability of the mitochondrial membrane. Upon apoptotic stimulation, Bax, a pore-forming cytoplasmic protein, translocates to the outer mitochondrial membrane, changes its permeability, and induces cytochrome c release from the mitochondria to the cytosol. The antiapoptotic Bcl-2 is associated with the outer mitochondrial membrane where it stabilizes the membrane permeability and thus suppresses the release of cytochrome c.26,35,36 During high glucose-induced apoptosis, a decrease of mitochondrial Bcl-2 expression and distribution of Bax from the cytosol to mitochondria were

significantly ameliorated by PSG-1, as evidenced by enhancing the activity of mitochondrial dehydrogenase and maintaining ΔΨm, indicating that PSG-1 may improve mitochondrial function in endothelial cells under high glucose conditions. In response to overproduction of ROS, the outer mitochondrial membrane becomes permeable, which leads to the loss of ΔΨm, triggers the release of cytochrome c from mitochondria to the cytoplasm, and activates the mitochondrial apoptotic pathway.35 It was consistent therefore that there was a significant decrease in ΔΨm in high glucose conditions in endothelial cells. We also found that release of cytochrome c from mitochondria to the cytoplasm was significantly increased. Incubation of endothelial cells with PSG-1 for 1 h significantly attenuated the effect of high glucose on the loss of ΔΨm and release of mitochondrial cytochrome c. The Bcl-2 family, which H


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candidate as a new interventional approach for diabetic angiopathy.

observed in endothelial cells. Significantly, the PSG-1 treatment decreased the alteration of Bcl-2 family protein expression in the endothelial cells stimulated by high glucose. The Bcl-2 family dependent changes in the permeability of the mitochondrial membrane that resulted in the loss of ΔΨm and the release of cytochrome c are mediated by a highconductance channel, mPTP. Although Hunter and Haworth in the late 1970s first observed and described mPTP, its function in the vascular complication of diabetes has still not been clarified.18,37−39 The present study was designed to further explore the role of mPTP in high glucose-induced apoptotic cell death. Our results not only supported the idea of an increase in the opening of mPTP as one of the key events during apoptotic cell death in high glucose but also suggested that mPTP may be an oxidative stress-sensitive channel opened by increased ROS production. Therefore, we have investigated whether inhibition of the opening of mPTP may prevent apoptotic cell death under high glucose conditions. Treatment with CsA significantly inhibited cell death under high glucose conditions, which supported the view that opening of mPTP may play a critical role in high glucose inducing cell death. PSG-1 also inhibited the opening of mPTP with an efficacy similar to that of CsA. PSG-1 can thus influence the oxidative stress-related opening of mPTP, which points to novel insight into the potential therapeutic targets for PSG-1 intervention. During transduction of an apoptotic signal into the mitochondria, the apoptotic protein of cytochrome c is translocated into the cytoplasm, which leads to the activation of caspase-9. Active caspase-9 then activates caspase-3, which contributes to the execution of apoptosis.40−42 In keeping with these observations, our results showed that high glucoseinduced apoptosis was accompanied by a significant increase of caspase-9 and caspase-3 activities, compared with cells under control conditions. Likewise, PSG-1 treatment significantly decreased the high glucose-induced activation of both caspases. Since the final pathway that contributes to the execution of cell death signal is caspase activation, we used a caspase inhibitor ZVAD-FMK to treat the endothelial cells under high glucose conditions. We found that the caspase inhibitor significantly decreases high glucose-stimulated apoptosis, which suggested that high glucose-induced apoptosis in the endothelial cells might be caspase-dependent, which could be alleviated by PSG1. Overall, we have provided in vitro evidence about the effects of the Ganoderma atrum polysaccharide, PSG-1, on diabetic angiopathy. These interesting findings suggested that PSG-1 alleviated HG-associated cell dysfunction by its effect on the modification of the redox system and implied that antioxidant effects of PSG-1 might be related to its protective effect against vascular dysfunction. On the basis of these results, we have drawn up a schematic model (Figure 6) for the effects of PSG-1 on endothelial cells under high glucose conditions, which describe how ROS production and the opening of mPTP are centrally involved in apoptosis and diabetic angiopathy. Accordingly, PSG-1 could ameliorate endothelial cell damage in response to high glucose via a mitochondria-reactive oxygen species pathway. Meanwhile, factors affecting the permeability of the mitochondrial membrane have a profound influence on cell survival and cell death. Future work is necessary to investigate the molecular basis of the relationship between mPTP and the health benefits of PSG-1 against diabetic angiopathy. Together, these findings make PSG-1 an attractive

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AUTHOR INFORMATION

Corresponding Author

*(M.-Y.X.) Tel: +86 791-3969009. Fax: +86 791-3969009. Email: [email protected]/[email protected]. Funding

The financial support for the present study by the National Natural Science Foundation of China (Nos: 31130041, 31471702, and 31201326), Natural Science Foundation of Jiangxi Province (20132BAB214001 and 20151BAB204038), and Key Science and Technique Project of Department of Science, Jiangxi Province, is gratefully acknowledged. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED CAD, caspase-activated deoxyribonuclease; CsA, cyclosporine A; DCF, 2′,7′-dichlorofluorescein; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; HUVECs, human umbilical vein cells; ICAD, inhibitor of caspase-activated deoxyribonuclease; IMS, intermembrane space; IMM, inner mitochondrial membrane; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide; LDH, lactate dehydrogenase; ΔΨm, mitochondrial membrane potential; mPTP, mitochondrial permeability transition pore; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim bromide; NAC, N-acetyl cysteine; OMM, outer mitochondrial membrane; PSG-1, Ganoderma atrum polysaccharide; ROS, reactive oxygen species; tBH, tert-butyl hydroperoxide

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REFERENCES

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Ganoderma atrum Polysaccharide Ameliorates Hyperglycemia

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