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Journal Pre-proof Ginsenoside Rb1 mitigates oxidative stress and apoptosis induced by methylglyoxal in SH-SY5Y cells via the PI3K/Akt pathway Fengwei Nan, Guibo Sun, Weijie Xie, Tianyuan Ye, Xiao Sun, Ping Zhou, Xi Dong, Jiafu Sun, Mengren Zhang, Xiaobo Sun PII:
S0890-8508(19)30299-3
DOI:
https://doi.org/10.1016/j.mcp.2019.101469
Reference:
YMCPR 101469
To appear in:
Molecular and Cellular Probes
Received Date: 9 August 2019 Revised Date:
26 September 2019
Accepted Date: 13 October 2019
Please cite this article as: Nan F, Sun G, Xie W, Ye T, Sun X, Zhou P, Dong X, Sun J, Zhang M, Sun X, Ginsenoside Rb1 mitigates oxidative stress and apoptosis induced by methylglyoxal in SH-SY5Y cells via the PI3K/Akt pathway, Molecular and Cellular Probes (2019), doi: https://doi.org/10.1016/ j.mcp.2019.101469. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Ginsenoside Rb1 Mitigates Oxidative Stress and Apoptosis Induced by Methylglyoxal in SH-SY5Y cells via the PI3K/Akt Pathway Fengwei Nana, Guibo Sunb, Weijie Xieb, Tianyuan Yeb, Xiao Sunb, Ping Zhoub, Xi Dongb, Jiafu Sunb, Mengren Zhanga,*, Xiaobo Sunb,** a
Department of Traditional Chinese Medicine, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, China b Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural Medicine) and Translational Medicine, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences,Beijing 100193, China *Corresponding author. Professor Mengren Zhang, Department of Traditional Chinese Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1 Shuaifuyuan, Dongcheng, Beijing 100730, P.R. China. E-mail address:[email protected] **Corresponding author. Professor Xiaobo Sun, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 151, Malianwa North Road, Haidian, Beijing 100193, P.R. China. E-mail addresses: [email protected] (X. Sun). Abstract Diabetic encephalopathy is a severe diabetic complication characterized by cognitive dysfunction and neuropsychiatric disability. Methylglyoxal (MGO), a highly reactive metabolite of hyperglycemia, serves as a major precursor of advanced glycation end products that play key roles in diabetic complications. Ginsenoside Rb1 (abbreviated as Rb1) has received extensive attention due to its potential therapeutic effects on diabetes and neurodegeneration. Therefore, this study aimed to investigate the effects of Rb1 on MGO-induced damage in SH-SY5Y cells and the related mechanism. SH-SY5Y cells were pretreated with Rb1 for 8 h and then exposed to MGO (0.5 mM) for 24 h. Cell survival was assessed by the MTT assay. Cell apoptosis was assessed using Hoechst 33342/propidium iodide (PI) staining and an Annexin-V/PI kit. The activities of oxidative stress markers were examined using commercial kits. Reactive oxygen species (ROS) staining and JC-1 staining were used to evaluate mitochondria injury. In addition, protein levels were measured by Western blot analysis. As a result, Rb1 alleviated the injury induced by MGO by increasing the activities of superoxide dismutase, catalase and total glutathione, decreasing the level of malondialdehyde,
and alleviating mitochondrial damage and ROS production. Furthermore, Rb1 could enhance the Bcl-2/Bax ratio, inhibit the expression of cleaved caspase-3 and cleaved caspase-9, and enhance the levels of phosphorylated Akt. Moreover, the protective effects of Rb1 against MGO-induced apoptosis were partly abolished by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) phosphorylation. Our results demonstrated that Rb1 ameliorated MGO-induced oxidative stress and apoptosis in SH-SY5Y cells via activating the PI3K/Akt signaling pathway. Keywords: ginsenoside Rb1; oxidative stress; apoptosis; methyglyoxal
1. Introduction Recently, diabetic encephalopathy (DE) has been recognized as a severe diabetic complication [1]. The main manifestations of DE are degenerative neurochemical, electrophysiological and structural changes in the brain and cognitive decline [2, 3]. In the brain of individuals with diabetes, hyperglycemic conditions and impaired glucose metabolism lead to abundant production of reactive carbonyl compounds, such as methylglyoxal (MGO) [4]. MGO, an intermediary degradation product of Maillard reactions, is produced by both enzymatic and nonenzymatic processes, including glycolysis, polyol pathways and threonine catabolism. It has been found that MGO levels are significantly elevated in patients with type 2 diabetes (T2DM) and Alzheimer’s disease (AD) [5-7]. Accumulated MGO binds with DNA, proteins, and lipids, leading to their oxidative modification and disturbances in their molecular functions. The cytotoxicity of MGO is mainly mediated through oxidative stress, further leading to apoptosis. In particular, MGO is a major potent precursor of advanced glycation end products (AGEs), which have also been implicated in the development of diabetic complications [8, 9]. In addition, the production of mitochondrial-derived reactive oxygen species (ROS) during MGO metabolism has also been extensively demonstrated. It is worth mentioning that some researchers believe that MGO-induced damage might resemble T2DM-induced complications more closely [9]. Moreover, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway is an important signal transduction pathway that is closely associated with cell proliferation, apoptosis and oxidative stress. After PI3K is activated, phosphatidyl inositol 3, 4, 5-triphosphate (PIP3) will combine with Akt in the cytoplasm. Subsequently, Akt will be phosphorylated and then play an anti-apoptosis role via regulating the expression of Bcl-2, Bax and caspase-3 [10, 11], which are associated with diabetes and its complications. Ginsenoside Rb1 (abbreviated as Rb1) is one of the major bioactive ingredients in Panax ginseng C.A. Meyer (Araliaceae), a prominent traditional Chinese herb commonly used for the clinical treatment of diabetes. Rb1 can enhance intelligence in normal animals and improve cognitive function in animals with ischemia, cognitive impairment, and diabetes [12, 13]. In vitro, Rb1 can increase Schwann cell proliferation and increase the secretion of nerve growth factor and brain-derived neurotrophic factor [12]. Our previous study also found that Rb1 protected primary hippocampal neurons from high glucose-induced injury by inhibiting endoplasmic
reticulum stress-related apoptosis signals and reducing intracellular ROS production [14, 15]. However, whether Rb1 can improve the neuronal injury caused by MGO remains unclear. Therefore, the present study aimed to investigate the effects of Rb1 on MGO-induced SH-SY5Y cells, especially focusing on the molecular mechanism in regard to oxidative stress and apoptosis. 2. Materials and methods 2.1 Reagents MGO and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rb1 (purity ≥ 98%) was obtained from Shanghai Winherb Medical Technology Co., Ltd. (Shanghai, China), and its structure is illustrated in Fig. 1. Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS) and penicillin streptomycin (Pen Strep) were provided by Gibco (Grand Island, NY, USA). The Annexin V/propidium iodide (PI) double-staining kit for flow cytometry was acquired from Invitrogen (Carlsbad, CA, USA). Detection kits for the activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and catalase (CAT) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) were obtained from Solarbio (Beijing, China). The fluorescent dye (JC-1) kits and assay kits for MDA and total glutathione (GSH) were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Anti-Bcl-2 and anti-Bax primary antibodies were obtained from Proteintech (Rosemont, IL, USA), and all other antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The specific PI3K inhibitor LY294002 was purchased from MedChemExpress (Monmouth, NJ, USA). Other chemicals were purchased from CoWin Biosciences (Beijing, China).
Fig. 1 The structure of Rb1. 2.2 Cell culture and treatment SH-SY5Y cells were obtained from the Cell Bank of the Chinese Academy of Medical Sciences (Beijing, China) and cultured in high glucose DMEM supplemented
with 10% FBS and 1% Pen Strep. The cells were maintained in a humidified incubator containing 5% CO2 at 37 °C. The cells were seeded at an appropriate density according to the experimental protocol and used for experiments at approximately 80% confluence. The grouping design was as follows: control group, Rb1 group, MGO group and MGO + Rb1 group. Besides, to further elucidate the role of the PI3K/Akt pathway on the effects of Rb1, the cells were pretreated with LY294002 (10 µM) for 1h before Rb1 was added. 2.3 Cell viability measurement and LDH assay Cell viability was measured by MTT assay. Briefly, SH-SY5Y cells were seeded in a 96-well plate with 3,000 cells/well and cultured overnight. After the supernatant was removed, the cells were incubated with various concentrations of MGO (0.125, 0.25, 0.5, 0.75, 1, and 2 mM) for 24 h to determine the optimal model conditions and the IC50 of MGO. Subsequently, 100 µL of MTT solution (1 mg/mL) was added to each well. After incubation at 37 °C for an additional 4 h, the supernatant was removed, and the formazan crystals were dissolved in 150 µL of DMSO. The absorbance was measured at 570 nm by a microplate reader (Biotek, Winooski, VT). After the modeling condition was confirmed, the cells were pretreated with different concentrations of Rb1 (0.375, 0.75, and 1.5 µM) for 8 h, followed by treatment with MGO for 24 h. Then, the cell viability was assayed. The LDH activity in the culture supernatant was measured according to the manufacturer’s instructions. 2.4 Flow Cytometry detection of apoptosis Annexin V-FITC/PI double-staining kits were used for the determination of cell apoptosis. Briefly, SH-SY5Y cells were cultured in 6-well collagen-coated plates and pretreated with 1.5 µM Rb1 for 8 h, followed by treatment with 0.5 mM MGO for 24 h. Subsequently, the cells were harvested and washed twice with precooled phosphate buffered saline (PBS) and then resuspended in 1× Annexin V binding buffer. Then, aliquots of 105 cells were mixed with 5 µL of the Annexin V-FITC working solution. After incubation in the dark for 15 min at room temperature (RT), 400 µL of 1× Annexin V binding buffer and 5 µL of PI were added into each tube. The preparations were analyzed by a FACSCalibur flow cytometer (BD, San Diego, CA). 2.5 Determination of mitochondrial transmembrane potential ∆Ψm (MMP) The fluorescent, lipophilic and cationic probe JC-1 was employed to measure the changes in MMP. Briefly, SH-SY5Y cells were cultured in 48-well plates and subjected to drug treatment as described above. After the supernatant was discarded, the cells were washed twice with PBS and then incubated with JC-1 staining solution (5 µg/mL) in the dark at 37 °C for 30 min. After washing with PBS, images were acquired by using a high-content imaging system Image Xpress Micro (Molecular Devices, USA). 2.6 MDA, SOD, CAT and total GSH measurements After the SH-SY5Y cells were treated with the specific drug and vehicle mentioned above, the content of MDA and the activities of intracellular SOD, CAT and total GSH were determined with the corresponding commercial kits according to the manufacturer’s instructions. 2.7 Hoechst 33342 and PI double staining
After treatment, the cells were washed with PBS. One hundred microliters of Hoechst 33342 staining solution (10 mg/mL) was added into each well, and the plate was incubated in the dark at 37 °C for 15 min, followed by the addition of 100 µL PI (100 mg/mL). After washing with PBS, images were acquired by using a high-content imaging system Image Xpress Micro (Molecular Devices, USA). 2.8 Intracellular reactive oxygen species (ROS) measurement The fluorescent probe DCFH-DA,, which could be oxidized to the highly fluorescent compound dichlorofluorscein (DCF), was used to measure the intracellular ROS. The cells were pretreated as mentioned above and then incubated with 10 µM DCFH-DA in the dark at 37 °C for 30 min. After the cells were washed twice with PBS, the cellular fluorescence intensity was quantified by using a high-content imaging system Image Xpres Micro (Molecular Devices, USA). 2.9 Western blot analysis After treatment, the cells were harvested and the whole-cell lysates protein was extracted by RIPA buffer containing 1% phosphatase inhibitor cocktail and 1% protease inhibitor cocktail (CoWin Bioscience Co., Ltd., Beijing, China). The protein concentration was measured by a BCA protein assay kit. A total of 20 µg protein was separated by 8% or 10% sodium dodecyl sulfate polyacrylamide gels (SDS–PAGE), and then was transferred onto nitrocellulose membranes. The membranes were blocked by 5% skim milk (dissolved in TBST) at RT for 2 h, and subsequently incubated with specific primary antibodies at 4 °C overnight. After washing with TBST three times, the bands were incubated with HRP-conjugated secondary antibodies at RT for 2 h. The proteins were visualized by electrochemiluminesence (ECL) kits and scanned with a ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA). The grayscale values of the visualized proteins were quantified by Bio-Rad Laboratories software (Hercules, CA, USA). 2.10 Statistical Analysis All data are expressed as the mean ± standard deviation (SD) with at least three independent experiments. One-way analysis of variance (ANOVA) followed by a Student-Newman-Keuls test was used to evaluate the differences among groups by GraphPad Prism 5.0. P < 0.05 was considered as statistical significance. 3. Results 3.1 Cytotoxicity of MGO on SH-SY5Y cells The cytotoxic effects of MGO at different concentrations (0.125, 0.25, 0.5, 0.75, 1, and 2 mM) on SH-SY5Y cells are shown in Fig. 2A. MGO significantly suppressed cell viability in a dose-dependent manner. Cell viability was maintained at approximately 60% when the concentration of MGO was set to 0.5 mM. According to the dose-effect curve of MGO (Fig. 2B), the 24 h IC50 value of MGO for SH-SY5Y cells was calculated to be 0.71 mM. Based on these results, 0.5 mM MGO for 24 h was selected as the optimal modeling condition for further study. 3.2 Cytoprotective effects of Rb1 on MGO-induced cell injury As shown in Fig. 2C, Rb1 had no significant impact on cell survival within 6 mM, while Rb1 at 12 mM markedly promoted cellular proliferation. Rb1 (0.375, 0.75,
and 1.5 µM) could reverse the decreased cell viability induced by MGO in a dose-dependent manner, and 1.5 µM Rb1 was the most effective dose (Fig. 2D). To further determine the optimal concentration of Rb1, LDH assays (Fig. 2E) were performed. LDH levels were significantly increased by MGO, while Rb1 could inhibit the LDH release in a dose-dependent manner, and 1.5 µM Rb1 was the most effective dose without cytotoxicity. Therefore, 1.5 µM Rb1 was used for further study.
Fig. 2 The protective effects of Rb1 on MGO-induced injury in SH-SY5Y cells. (A) The effects of MGO at various concentrations on cell viability. (B) The dose-effect curve of MGO. (C) The toxic effects of Rb1 on cell viability. (D) The effects of Rb1 on cell viability induced by MGO. SH-SY5Y cells were pretreated with Rb1 (0.375, 0.75, and 1.5 µM) for 8 h, and then incubated with MGO (0.5 mM) for 24 h. (E) The effects of Rb1 on LDH release. (means ± SD, n = 6). ##p < 0.01 vs. control group; *p < 0.05, **p < 0.01 vs. MGO group. 3.3 Rb1 ameliorated MGO-induced oxidative stress in SH-SY5Y cells. The probe DCFH-DA could be oxidized into the fluorescent compound DCF, which indirectly reflected the level of ROS in SH-SY5Y cells. As shown in Fig. 3A and 3B, the DCF fluorescence intensity was distinctly strengthened by MGO, indicating the excessive accumulation of ROS. Rb1 could weaken the intracellular ROS level and suppress the oxidative stress induced by MGO. Furthermore, lipid peroxide and antioxidant enzymes and substances were examined by using the respective kits. The content of MDA was obviously increased (Fig. 3C), while the levels of SOD, CAT and total GSH (Fig. 3D, 3E and 3F) were significantly decreased in the MGO group compared to the control group. Rb1 treatment corrected the imbalance between the oxidation and antioxidation systems in the SH-SY5Y cells.
Fig. 3 Rb1 suppressed MGO-induced oxidative stress in SH-SY5Y cells. SH-SY5Y cells were pretreated with Rb1 (1.5 µM) for 8 h, and then incubated with MGO (0.5 mM) for 24 h. (A) Representative photographs of ROS staining. (B) Quantitative analysis of the DCF fluorescence intensity. (C) The levels of MDA. (D) The levels of SOD. (E) The levels of CAT. (F) The levels of total GSH. (mean ± SD, n = 3). ##p < 0.01 vs. control group; **p < 0.01 vs. MGO group. 3.4 Rb1 attenuated the decline in MMP in SH-SY5Y cells In this study, the changes in MMP were measured by JC-1 staining (Fig. 4A). MGO caused a remarkable increase in the ratio of green to red, while Rb1 reduced the green fluorescence (Fig. 4B), suggesting that Rb1 could attenuate the mitochondrial damage induced by MGO in SH-SY5Y cells.
Fig. 4 Effects of Rb1 on mitochondrial transmembrane potential. SH-SY5Y cells were pretreated with Rb1 (1.5 µM) for 8 h, and then incubated with MGO (0.5 mM) for 24 h. (A) Representative photographs of the JC-1 staining. (B) Quantitative analysis of the ratio of green to red in JC-1 staining. (mean ± SD, n = 3). ##p < 0.01 vs. control group; **p < 0.01 vs. MGO group. 3.5 Rb1 reversed MGO-induced apoptosis in SH-SY5Y cells MGO induced an increase in condensed nuclei and PI positive cells in Hoechst 33342/PI staining, which could be reduced by Rb1 (Fig. 5A). Meanwhile, the level of apoptosis was detected by flow cytometry using Annexin V/PI staining (Fig. 5B). The percentage of apoptotic cells in the upper-right and lower-right quadrants were analyzed and are shown in Fig. 5C. MGO caused an increase in apoptosis, whereas Rb1 decreased apoptosis, indicating that Rb1 exerted cytoprotective effects by inhibiting apoptosis of SH-SY5Y cells.
Fig. 5 The effects of Rb1 on apoptosis induced by MGO in SH-SY5Y cells. SH-SY5Y cells were pretreated with Rb1 (1.5 µM) for 8 h and then incubated with MGO (0.5 mM) for 24 h. (A) Representative images of Hoechst33342/PI staining. (B) and (C) Apoptosis analysis quantified by flow cytometry. (mean ± SD, n = 3). ##p < 0.01 vs. control group; **p < 0.01 vs. MGO group. 3.6 The effects of Rb1 on apoptosis-related proteins Western blot analysis was performed to detect the expression of pro- and anti-apoptotic proteins in SH-SY5Y cells, as presented in Fig. 6. The levels of cleaved caspase-3 and cleaved caspase-9 were significantly upregulated, while the ratio of Bcl-2/Bax was remarkably downregulated in the MGO group compared to that in the control group. Rb1 treatment could alleviate the abnormalities of the apoptosis-related proteins induced by MGO, implying an anti-apoptosis effect of Rb1 on SH-SY5Y cells.
Fig. 6 The effects of Rb1 on apoptosis-related protein expression in SH-SY5Y cells. (A) The representative protein bands of Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9, and β-actin. (B) The ratio of Bcl-2/Bax. (C) The relative level of cleaved caspase-3. (D) The relative level of cleaved caspase-9. (mean ± SD, n = 3). ##p < 0.01 vs. control group; *p < 0.05, **p < 0.01 vs. MGO group. 3.7 The effects of Rb1 on the PI3K/Akt signaling pathway The PI3K/Akt pathway plays a key role in cell growth and participates in the inhibition of oxidative stress. The expression of Akt and p-Akt was measured by Western blot (Fig. 7). MGO significantly inhibited the level of p-Akt (Fig. 7B), while Rb1 administration remarkably increased the activation of Akt by phosphorylation. However, pretreatment of LY294002 significantly weakened the protective effect of Rb1 (Fig. 8). LY294002 obviously blocked Rb1-reversed cell viability (Fig. 8A) and cell apoptosis (Fig. 8D). Meanwhile, the Rb1-mediated phosphorylation of Akt was significantly decreased by LY294002 (Fig. 8B). These results indicated that Rb1 could activate the PI3K/Akt signaling pathway and thus protect SH-SY5Y cells from MGO-induced apoptosis and oxidative injury.
Fig. 7 Effects of Rb1 on the PI3K/Akt signaling pathway. (A) The representative protein bands of p-Akt and Akt. (B) The ratio of p-Akt/Akt. (mean ± SD, n = 3). ##p < 0.01 vs. control group; **p < 0.01 vs. MGO group.
Fig. 8 Effects of LY294002 (a specific inhibitor of PI3K, abbreviated as LY) on the protection of Rb1 against MGO-induced cell death and apoptosis. SH-SY5Y cells were pretreated with LY294002 (10μM) for 1 h, and then incubated with Rb1 (1.5μ M) for 8 h before the treatment of MGO. (A) The effects of LY294002 on cell viability (means ± SD, n = 6). (B) and (C) The representative protein of p-Akt and Akt, and the ratio of p-Akt/Akt. (D) and (E) Apoptosis analysis quantified by flow cytometry. (mean ± SD, n = 3). ##p < 0.01 vs. control group; *p < 0.05, **p < 0.01 vs. MGO group; $p < 0.05, $$ p < 0.01 vs. MGO+Rb1 group. 4. Discussion DE, a disorder occurring in the brain of diabetes patients, is recognized as a severe complication of diabetes [2, 16]. Hyperglycemia has been considered the primary pathogenic factor of diabetic complications for a long time. In recent years, however, a large number of clinical trials have demonstrated that even when HbA1c can be maintained at ≤6.5% by hypoglycemic drugs, the progression of typical diabetes complications could not be suppressed [17, 18]. This observation suggests that there might be other mechanisms in addition to high glucose-induced tissue
damage in diabetes. One of the putative causes is the formation of MGO. Epidemiological studies have revealed that T2DM accounts for approximately 90% of diabetes cases. As the most reactive precursor of AGEs, MGO is increased in T2DM patients and is linked to diabetic complications [19]. Researchers found that MGO accumulation in glyoxalase 1 (Glo1) knockout Drosophila resulted in insulin resistance, obesity and hyperglycemia. Glo1 is one of the major lytic enzymes of MGO. This finding proved that MGO is not the result, but may instead be the cause, of T2DM [17], which is the converse of previous conceptions. In addition, elevated MGO promoted protein glycation and oxidative stress, contributing to diabetes-associated cognitive decline in rats [9, 20]. Inevitably, the elevated serum concentration of MGO in elderly individuals is related to increased cognitive decline [21]. Therefore, MGO was selected in this study as the inducer to injure SH-SY5Y cells, simulating the neuron damage in DE. We observed that Rb1 had a neuroprotective effect on MGO-induced neurotoxicity as displayed by the alleviation of the decreased cell viability and increased neuronal apoptosis of the SH-SY5Y cells. The mechanisms of its neuroprotective effect may be involved in the inhibition of oxidative stress-related apoptosis. Although the pathogenesis of DE has not been clearly elucidated, protein glycation, oxidative stress and neuron apoptosis are significantly increased in the brain during this condition. Under physiological conditions, the organism will produce ROS and superoxide anion radicals constantly, most of which will be scavenged by the antioxidant enzyme system, including SOD, glutathione peroxidase (GSH-Px), and CAT. However, excessive production of oxygen free radicals together with antioxidant deficiency in the brain causes pathologic changes that are a risk factor for DE [22, 23]. In primary hippocampal neurons, MGO could increase oxidative stress, reduce the activity of antioxidant enzymes, and further induce apoptosis, which explains the possible mechanism of MGO-induced DE [24, 25]. Consistent with these reports, our results also showed that MGO upregulated the level of MDA and downregulated the contents of SOD, CAT, and total GSH, which was markedly reversed by Rb1. These results suggest that the protective effects of Rb1 on MGO-induced SH-SY5Y injury are mediated through inhibiting oxidative stress. In agreement with a former study [26], this experiment proved that MGO increased the generation of ROS, which could dampen the mitochondrial antioxidant defense system and cause oxidative damage [27]. MGO can trigger abnormal opening of the mitochondrial transition pore by the collapse of MMP [28]. As a result, the outer membrane of the mitochondria releases cytochrome C, which recruits its receptor apoptotic protease activating factor 1, ATP, and pro-caspase-9 in the cytoplasm to form an apoptosome. Subsequently, pro-caspase-9 is activated and transformed to caspase-9, and pro-caspase-3 is cleaved to active caspase-3, leading to apoptosis [29]. In contrast, the anti-apoptotic protein Bcl-2, residing in the outer mitochondrial membrane, can form complexes with the pro-apoptosis protein Bax to inhibit the apoptosis induced by mitochondrial damage [30]. Bcl-2 also has a neuroprotective function in the brain [31]. Our results showed that MGO caused the increase in ROS levels and the decrease in MMP, while Rb1 decreased the ROS
generation and alleviated mitochondria dysfunction. From the Hoechst 33342/PI and Annexin V/PI staining, we found MGO caused an elevation of both early stage and later stage apoptosis, and Rb1 treatment significantly reduced the detrimental effects of MGO. Meanwhile, the expression of cleaved caspase-9 and cleaved caspase-3 were increased, and the Bcl-2/Bax ratio was decreased after MGO incubation, and these effects were all suppressed by Rb1. These results suggest that Rb1 could protect SH-SY5Y cells from MGO-induced injury through inhibiting ROS and apoptosis. The PI3K/Akt signaling pathway can regulate the survival, migration, metabolism, and proliferation of cells. Considerable studies have also illustrated that the activation of the PI3K/Akt signaling pathway is involved in anti-apoptosis and antioxidative stress in DE [32, 33]. As a major downstream effector protein of PI3K, Akt directly inhibits pro-apoptosis signals, including Bax [34], to protect neurons. Therefore, we identified the protein expression of Akt and level of phosphorylated Akt (p-Akt). The results showed that MGO inhibited the activation of the PI3K/Akt signaling pathway in SH-SY5Y cells, and Rb1 could reverse this alteration. In addition, inhibition of PI3K/Akt signaling partly abolished the cytoprotective effect of Rb1. Taken together, these results demonstrated that Rb1 enhanced the antioxidative stress and anti-apoptosis capacity of SH-SY5Y cells by activating the PI3K/Akt signaling pathway. 5. Conclusion Taken together, the present study revealed that ginsenoside Rb1 effectively protects SH-SY5Y cells from MGO-induced oxidative stress and apoptosis. The mechanism is probably through the activation of the PI3K/Akt signaling pathway. These findings may provide new avenues of research for the prevention and treatment of DE. Acknowledgements This research was supported by the Major Program of the National Natural Science Foundation of China (NO. 81891012). The authors have declared that no competing interests exist. In addition, Fengwei Nan wants to thank Yunfeng Zhou for his loyalty, his patience, and his love all these years. References [1]
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Highlights Methylglyoxal plays a key role in diabetic encephalopathy, and it exerts a detrimental effect featured by oxidative stress and apoptosis on SH-SY5Y cells. Ginsenoside Rb1 protected SH-SY5Y cells from methylglyoxal-induced oxidative stress and apoptosis. The neuroprotective mechanisms of ginsenoside Rb1 might be involved in regulating the PI3K/Akt signaling pathway.
S0890-8508(19)30299-3
DOI:
https://doi.org/10.1016/j.mcp.2019.101469
Reference:
YMCPR 101469
To appear in:
Molecular and Cellular Probes
Received Date: 9 August 2019 Revised Date:
26 September 2019
Accepted Date: 13 October 2019
Please cite this article as: Nan F, Sun G, Xie W, Ye T, Sun X, Zhou P, Dong X, Sun J, Zhang M, Sun X, Ginsenoside Rb1 mitigates oxidative stress and apoptosis induced by methylglyoxal in SH-SY5Y cells via the PI3K/Akt pathway, Molecular and Cellular Probes (2019), doi: https://doi.org/10.1016/ j.mcp.2019.101469. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Ginsenoside Rb1 Mitigates Oxidative Stress and Apoptosis Induced by Methylglyoxal in SH-SY5Y cells via the PI3K/Akt Pathway Fengwei Nana, Guibo Sunb, Weijie Xieb, Tianyuan Yeb, Xiao Sunb, Ping Zhoub, Xi Dongb, Jiafu Sunb, Mengren Zhanga,*, Xiaobo Sunb,** a
Department of Traditional Chinese Medicine, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, China b Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural Medicine) and Translational Medicine, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences,Beijing 100193, China *Corresponding author. Professor Mengren Zhang, Department of Traditional Chinese Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1 Shuaifuyuan, Dongcheng, Beijing 100730, P.R. China. E-mail address:[email protected] **Corresponding author. Professor Xiaobo Sun, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 151, Malianwa North Road, Haidian, Beijing 100193, P.R. China. E-mail addresses: [email protected] (X. Sun). Abstract Diabetic encephalopathy is a severe diabetic complication characterized by cognitive dysfunction and neuropsychiatric disability. Methylglyoxal (MGO), a highly reactive metabolite of hyperglycemia, serves as a major precursor of advanced glycation end products that play key roles in diabetic complications. Ginsenoside Rb1 (abbreviated as Rb1) has received extensive attention due to its potential therapeutic effects on diabetes and neurodegeneration. Therefore, this study aimed to investigate the effects of Rb1 on MGO-induced damage in SH-SY5Y cells and the related mechanism. SH-SY5Y cells were pretreated with Rb1 for 8 h and then exposed to MGO (0.5 mM) for 24 h. Cell survival was assessed by the MTT assay. Cell apoptosis was assessed using Hoechst 33342/propidium iodide (PI) staining and an Annexin-V/PI kit. The activities of oxidative stress markers were examined using commercial kits. Reactive oxygen species (ROS) staining and JC-1 staining were used to evaluate mitochondria injury. In addition, protein levels were measured by Western blot analysis. As a result, Rb1 alleviated the injury induced by MGO by increasing the activities of superoxide dismutase, catalase and total glutathione, decreasing the level of malondialdehyde,
and alleviating mitochondrial damage and ROS production. Furthermore, Rb1 could enhance the Bcl-2/Bax ratio, inhibit the expression of cleaved caspase-3 and cleaved caspase-9, and enhance the levels of phosphorylated Akt. Moreover, the protective effects of Rb1 against MGO-induced apoptosis were partly abolished by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) phosphorylation. Our results demonstrated that Rb1 ameliorated MGO-induced oxidative stress and apoptosis in SH-SY5Y cells via activating the PI3K/Akt signaling pathway. Keywords: ginsenoside Rb1; oxidative stress; apoptosis; methyglyoxal
1. Introduction Recently, diabetic encephalopathy (DE) has been recognized as a severe diabetic complication [1]. The main manifestations of DE are degenerative neurochemical, electrophysiological and structural changes in the brain and cognitive decline [2, 3]. In the brain of individuals with diabetes, hyperglycemic conditions and impaired glucose metabolism lead to abundant production of reactive carbonyl compounds, such as methylglyoxal (MGO) [4]. MGO, an intermediary degradation product of Maillard reactions, is produced by both enzymatic and nonenzymatic processes, including glycolysis, polyol pathways and threonine catabolism. It has been found that MGO levels are significantly elevated in patients with type 2 diabetes (T2DM) and Alzheimer’s disease (AD) [5-7]. Accumulated MGO binds with DNA, proteins, and lipids, leading to their oxidative modification and disturbances in their molecular functions. The cytotoxicity of MGO is mainly mediated through oxidative stress, further leading to apoptosis. In particular, MGO is a major potent precursor of advanced glycation end products (AGEs), which have also been implicated in the development of diabetic complications [8, 9]. In addition, the production of mitochondrial-derived reactive oxygen species (ROS) during MGO metabolism has also been extensively demonstrated. It is worth mentioning that some researchers believe that MGO-induced damage might resemble T2DM-induced complications more closely [9]. Moreover, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway is an important signal transduction pathway that is closely associated with cell proliferation, apoptosis and oxidative stress. After PI3K is activated, phosphatidyl inositol 3, 4, 5-triphosphate (PIP3) will combine with Akt in the cytoplasm. Subsequently, Akt will be phosphorylated and then play an anti-apoptosis role via regulating the expression of Bcl-2, Bax and caspase-3 [10, 11], which are associated with diabetes and its complications. Ginsenoside Rb1 (abbreviated as Rb1) is one of the major bioactive ingredients in Panax ginseng C.A. Meyer (Araliaceae), a prominent traditional Chinese herb commonly used for the clinical treatment of diabetes. Rb1 can enhance intelligence in normal animals and improve cognitive function in animals with ischemia, cognitive impairment, and diabetes [12, 13]. In vitro, Rb1 can increase Schwann cell proliferation and increase the secretion of nerve growth factor and brain-derived neurotrophic factor [12]. Our previous study also found that Rb1 protected primary hippocampal neurons from high glucose-induced injury by inhibiting endoplasmic
reticulum stress-related apoptosis signals and reducing intracellular ROS production [14, 15]. However, whether Rb1 can improve the neuronal injury caused by MGO remains unclear. Therefore, the present study aimed to investigate the effects of Rb1 on MGO-induced SH-SY5Y cells, especially focusing on the molecular mechanism in regard to oxidative stress and apoptosis. 2. Materials and methods 2.1 Reagents MGO and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rb1 (purity ≥ 98%) was obtained from Shanghai Winherb Medical Technology Co., Ltd. (Shanghai, China), and its structure is illustrated in Fig. 1. Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS) and penicillin streptomycin (Pen Strep) were provided by Gibco (Grand Island, NY, USA). The Annexin V/propidium iodide (PI) double-staining kit for flow cytometry was acquired from Invitrogen (Carlsbad, CA, USA). Detection kits for the activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and catalase (CAT) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) were obtained from Solarbio (Beijing, China). The fluorescent dye (JC-1) kits and assay kits for MDA and total glutathione (GSH) were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Anti-Bcl-2 and anti-Bax primary antibodies were obtained from Proteintech (Rosemont, IL, USA), and all other antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The specific PI3K inhibitor LY294002 was purchased from MedChemExpress (Monmouth, NJ, USA). Other chemicals were purchased from CoWin Biosciences (Beijing, China).
Fig. 1 The structure of Rb1. 2.2 Cell culture and treatment SH-SY5Y cells were obtained from the Cell Bank of the Chinese Academy of Medical Sciences (Beijing, China) and cultured in high glucose DMEM supplemented
with 10% FBS and 1% Pen Strep. The cells were maintained in a humidified incubator containing 5% CO2 at 37 °C. The cells were seeded at an appropriate density according to the experimental protocol and used for experiments at approximately 80% confluence. The grouping design was as follows: control group, Rb1 group, MGO group and MGO + Rb1 group. Besides, to further elucidate the role of the PI3K/Akt pathway on the effects of Rb1, the cells were pretreated with LY294002 (10 µM) for 1h before Rb1 was added. 2.3 Cell viability measurement and LDH assay Cell viability was measured by MTT assay. Briefly, SH-SY5Y cells were seeded in a 96-well plate with 3,000 cells/well and cultured overnight. After the supernatant was removed, the cells were incubated with various concentrations of MGO (0.125, 0.25, 0.5, 0.75, 1, and 2 mM) for 24 h to determine the optimal model conditions and the IC50 of MGO. Subsequently, 100 µL of MTT solution (1 mg/mL) was added to each well. After incubation at 37 °C for an additional 4 h, the supernatant was removed, and the formazan crystals were dissolved in 150 µL of DMSO. The absorbance was measured at 570 nm by a microplate reader (Biotek, Winooski, VT). After the modeling condition was confirmed, the cells were pretreated with different concentrations of Rb1 (0.375, 0.75, and 1.5 µM) for 8 h, followed by treatment with MGO for 24 h. Then, the cell viability was assayed. The LDH activity in the culture supernatant was measured according to the manufacturer’s instructions. 2.4 Flow Cytometry detection of apoptosis Annexin V-FITC/PI double-staining kits were used for the determination of cell apoptosis. Briefly, SH-SY5Y cells were cultured in 6-well collagen-coated plates and pretreated with 1.5 µM Rb1 for 8 h, followed by treatment with 0.5 mM MGO for 24 h. Subsequently, the cells were harvested and washed twice with precooled phosphate buffered saline (PBS) and then resuspended in 1× Annexin V binding buffer. Then, aliquots of 105 cells were mixed with 5 µL of the Annexin V-FITC working solution. After incubation in the dark for 15 min at room temperature (RT), 400 µL of 1× Annexin V binding buffer and 5 µL of PI were added into each tube. The preparations were analyzed by a FACSCalibur flow cytometer (BD, San Diego, CA). 2.5 Determination of mitochondrial transmembrane potential ∆Ψm (MMP) The fluorescent, lipophilic and cationic probe JC-1 was employed to measure the changes in MMP. Briefly, SH-SY5Y cells were cultured in 48-well plates and subjected to drug treatment as described above. After the supernatant was discarded, the cells were washed twice with PBS and then incubated with JC-1 staining solution (5 µg/mL) in the dark at 37 °C for 30 min. After washing with PBS, images were acquired by using a high-content imaging system Image Xpress Micro (Molecular Devices, USA). 2.6 MDA, SOD, CAT and total GSH measurements After the SH-SY5Y cells were treated with the specific drug and vehicle mentioned above, the content of MDA and the activities of intracellular SOD, CAT and total GSH were determined with the corresponding commercial kits according to the manufacturer’s instructions. 2.7 Hoechst 33342 and PI double staining
After treatment, the cells were washed with PBS. One hundred microliters of Hoechst 33342 staining solution (10 mg/mL) was added into each well, and the plate was incubated in the dark at 37 °C for 15 min, followed by the addition of 100 µL PI (100 mg/mL). After washing with PBS, images were acquired by using a high-content imaging system Image Xpress Micro (Molecular Devices, USA). 2.8 Intracellular reactive oxygen species (ROS) measurement The fluorescent probe DCFH-DA,, which could be oxidized to the highly fluorescent compound dichlorofluorscein (DCF), was used to measure the intracellular ROS. The cells were pretreated as mentioned above and then incubated with 10 µM DCFH-DA in the dark at 37 °C for 30 min. After the cells were washed twice with PBS, the cellular fluorescence intensity was quantified by using a high-content imaging system Image Xpres Micro (Molecular Devices, USA). 2.9 Western blot analysis After treatment, the cells were harvested and the whole-cell lysates protein was extracted by RIPA buffer containing 1% phosphatase inhibitor cocktail and 1% protease inhibitor cocktail (CoWin Bioscience Co., Ltd., Beijing, China). The protein concentration was measured by a BCA protein assay kit. A total of 20 µg protein was separated by 8% or 10% sodium dodecyl sulfate polyacrylamide gels (SDS–PAGE), and then was transferred onto nitrocellulose membranes. The membranes were blocked by 5% skim milk (dissolved in TBST) at RT for 2 h, and subsequently incubated with specific primary antibodies at 4 °C overnight. After washing with TBST three times, the bands were incubated with HRP-conjugated secondary antibodies at RT for 2 h. The proteins were visualized by electrochemiluminesence (ECL) kits and scanned with a ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA). The grayscale values of the visualized proteins were quantified by Bio-Rad Laboratories software (Hercules, CA, USA). 2.10 Statistical Analysis All data are expressed as the mean ± standard deviation (SD) with at least three independent experiments. One-way analysis of variance (ANOVA) followed by a Student-Newman-Keuls test was used to evaluate the differences among groups by GraphPad Prism 5.0. P < 0.05 was considered as statistical significance. 3. Results 3.1 Cytotoxicity of MGO on SH-SY5Y cells The cytotoxic effects of MGO at different concentrations (0.125, 0.25, 0.5, 0.75, 1, and 2 mM) on SH-SY5Y cells are shown in Fig. 2A. MGO significantly suppressed cell viability in a dose-dependent manner. Cell viability was maintained at approximately 60% when the concentration of MGO was set to 0.5 mM. According to the dose-effect curve of MGO (Fig. 2B), the 24 h IC50 value of MGO for SH-SY5Y cells was calculated to be 0.71 mM. Based on these results, 0.5 mM MGO for 24 h was selected as the optimal modeling condition for further study. 3.2 Cytoprotective effects of Rb1 on MGO-induced cell injury As shown in Fig. 2C, Rb1 had no significant impact on cell survival within 6 mM, while Rb1 at 12 mM markedly promoted cellular proliferation. Rb1 (0.375, 0.75,
and 1.5 µM) could reverse the decreased cell viability induced by MGO in a dose-dependent manner, and 1.5 µM Rb1 was the most effective dose (Fig. 2D). To further determine the optimal concentration of Rb1, LDH assays (Fig. 2E) were performed. LDH levels were significantly increased by MGO, while Rb1 could inhibit the LDH release in a dose-dependent manner, and 1.5 µM Rb1 was the most effective dose without cytotoxicity. Therefore, 1.5 µM Rb1 was used for further study.
Fig. 2 The protective effects of Rb1 on MGO-induced injury in SH-SY5Y cells. (A) The effects of MGO at various concentrations on cell viability. (B) The dose-effect curve of MGO. (C) The toxic effects of Rb1 on cell viability. (D) The effects of Rb1 on cell viability induced by MGO. SH-SY5Y cells were pretreated with Rb1 (0.375, 0.75, and 1.5 µM) for 8 h, and then incubated with MGO (0.5 mM) for 24 h. (E) The effects of Rb1 on LDH release. (means ± SD, n = 6). ##p < 0.01 vs. control group; *p < 0.05, **p < 0.01 vs. MGO group. 3.3 Rb1 ameliorated MGO-induced oxidative stress in SH-SY5Y cells. The probe DCFH-DA could be oxidized into the fluorescent compound DCF, which indirectly reflected the level of ROS in SH-SY5Y cells. As shown in Fig. 3A and 3B, the DCF fluorescence intensity was distinctly strengthened by MGO, indicating the excessive accumulation of ROS. Rb1 could weaken the intracellular ROS level and suppress the oxidative stress induced by MGO. Furthermore, lipid peroxide and antioxidant enzymes and substances were examined by using the respective kits. The content of MDA was obviously increased (Fig. 3C), while the levels of SOD, CAT and total GSH (Fig. 3D, 3E and 3F) were significantly decreased in the MGO group compared to the control group. Rb1 treatment corrected the imbalance between the oxidation and antioxidation systems in the SH-SY5Y cells.
Fig. 3 Rb1 suppressed MGO-induced oxidative stress in SH-SY5Y cells. SH-SY5Y cells were pretreated with Rb1 (1.5 µM) for 8 h, and then incubated with MGO (0.5 mM) for 24 h. (A) Representative photographs of ROS staining. (B) Quantitative analysis of the DCF fluorescence intensity. (C) The levels of MDA. (D) The levels of SOD. (E) The levels of CAT. (F) The levels of total GSH. (mean ± SD, n = 3). ##p < 0.01 vs. control group; **p < 0.01 vs. MGO group. 3.4 Rb1 attenuated the decline in MMP in SH-SY5Y cells In this study, the changes in MMP were measured by JC-1 staining (Fig. 4A). MGO caused a remarkable increase in the ratio of green to red, while Rb1 reduced the green fluorescence (Fig. 4B), suggesting that Rb1 could attenuate the mitochondrial damage induced by MGO in SH-SY5Y cells.
Fig. 4 Effects of Rb1 on mitochondrial transmembrane potential. SH-SY5Y cells were pretreated with Rb1 (1.5 µM) for 8 h, and then incubated with MGO (0.5 mM) for 24 h. (A) Representative photographs of the JC-1 staining. (B) Quantitative analysis of the ratio of green to red in JC-1 staining. (mean ± SD, n = 3). ##p < 0.01 vs. control group; **p < 0.01 vs. MGO group. 3.5 Rb1 reversed MGO-induced apoptosis in SH-SY5Y cells MGO induced an increase in condensed nuclei and PI positive cells in Hoechst 33342/PI staining, which could be reduced by Rb1 (Fig. 5A). Meanwhile, the level of apoptosis was detected by flow cytometry using Annexin V/PI staining (Fig. 5B). The percentage of apoptotic cells in the upper-right and lower-right quadrants were analyzed and are shown in Fig. 5C. MGO caused an increase in apoptosis, whereas Rb1 decreased apoptosis, indicating that Rb1 exerted cytoprotective effects by inhibiting apoptosis of SH-SY5Y cells.
Fig. 5 The effects of Rb1 on apoptosis induced by MGO in SH-SY5Y cells. SH-SY5Y cells were pretreated with Rb1 (1.5 µM) for 8 h and then incubated with MGO (0.5 mM) for 24 h. (A) Representative images of Hoechst33342/PI staining. (B) and (C) Apoptosis analysis quantified by flow cytometry. (mean ± SD, n = 3). ##p < 0.01 vs. control group; **p < 0.01 vs. MGO group. 3.6 The effects of Rb1 on apoptosis-related proteins Western blot analysis was performed to detect the expression of pro- and anti-apoptotic proteins in SH-SY5Y cells, as presented in Fig. 6. The levels of cleaved caspase-3 and cleaved caspase-9 were significantly upregulated, while the ratio of Bcl-2/Bax was remarkably downregulated in the MGO group compared to that in the control group. Rb1 treatment could alleviate the abnormalities of the apoptosis-related proteins induced by MGO, implying an anti-apoptosis effect of Rb1 on SH-SY5Y cells.
Fig. 6 The effects of Rb1 on apoptosis-related protein expression in SH-SY5Y cells. (A) The representative protein bands of Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9, and β-actin. (B) The ratio of Bcl-2/Bax. (C) The relative level of cleaved caspase-3. (D) The relative level of cleaved caspase-9. (mean ± SD, n = 3). ##p < 0.01 vs. control group; *p < 0.05, **p < 0.01 vs. MGO group. 3.7 The effects of Rb1 on the PI3K/Akt signaling pathway The PI3K/Akt pathway plays a key role in cell growth and participates in the inhibition of oxidative stress. The expression of Akt and p-Akt was measured by Western blot (Fig. 7). MGO significantly inhibited the level of p-Akt (Fig. 7B), while Rb1 administration remarkably increased the activation of Akt by phosphorylation. However, pretreatment of LY294002 significantly weakened the protective effect of Rb1 (Fig. 8). LY294002 obviously blocked Rb1-reversed cell viability (Fig. 8A) and cell apoptosis (Fig. 8D). Meanwhile, the Rb1-mediated phosphorylation of Akt was significantly decreased by LY294002 (Fig. 8B). These results indicated that Rb1 could activate the PI3K/Akt signaling pathway and thus protect SH-SY5Y cells from MGO-induced apoptosis and oxidative injury.
Fig. 7 Effects of Rb1 on the PI3K/Akt signaling pathway. (A) The representative protein bands of p-Akt and Akt. (B) The ratio of p-Akt/Akt. (mean ± SD, n = 3). ##p < 0.01 vs. control group; **p < 0.01 vs. MGO group.
Fig. 8 Effects of LY294002 (a specific inhibitor of PI3K, abbreviated as LY) on the protection of Rb1 against MGO-induced cell death and apoptosis. SH-SY5Y cells were pretreated with LY294002 (10μM) for 1 h, and then incubated with Rb1 (1.5μ M) for 8 h before the treatment of MGO. (A) The effects of LY294002 on cell viability (means ± SD, n = 6). (B) and (C) The representative protein of p-Akt and Akt, and the ratio of p-Akt/Akt. (D) and (E) Apoptosis analysis quantified by flow cytometry. (mean ± SD, n = 3). ##p < 0.01 vs. control group; *p < 0.05, **p < 0.01 vs. MGO group; $p < 0.05, $$ p < 0.01 vs. MGO+Rb1 group. 4. Discussion DE, a disorder occurring in the brain of diabetes patients, is recognized as a severe complication of diabetes [2, 16]. Hyperglycemia has been considered the primary pathogenic factor of diabetic complications for a long time. In recent years, however, a large number of clinical trials have demonstrated that even when HbA1c can be maintained at ≤6.5% by hypoglycemic drugs, the progression of typical diabetes complications could not be suppressed [17, 18]. This observation suggests that there might be other mechanisms in addition to high glucose-induced tissue
damage in diabetes. One of the putative causes is the formation of MGO. Epidemiological studies have revealed that T2DM accounts for approximately 90% of diabetes cases. As the most reactive precursor of AGEs, MGO is increased in T2DM patients and is linked to diabetic complications [19]. Researchers found that MGO accumulation in glyoxalase 1 (Glo1) knockout Drosophila resulted in insulin resistance, obesity and hyperglycemia. Glo1 is one of the major lytic enzymes of MGO. This finding proved that MGO is not the result, but may instead be the cause, of T2DM [17], which is the converse of previous conceptions. In addition, elevated MGO promoted protein glycation and oxidative stress, contributing to diabetes-associated cognitive decline in rats [9, 20]. Inevitably, the elevated serum concentration of MGO in elderly individuals is related to increased cognitive decline [21]. Therefore, MGO was selected in this study as the inducer to injure SH-SY5Y cells, simulating the neuron damage in DE. We observed that Rb1 had a neuroprotective effect on MGO-induced neurotoxicity as displayed by the alleviation of the decreased cell viability and increased neuronal apoptosis of the SH-SY5Y cells. The mechanisms of its neuroprotective effect may be involved in the inhibition of oxidative stress-related apoptosis. Although the pathogenesis of DE has not been clearly elucidated, protein glycation, oxidative stress and neuron apoptosis are significantly increased in the brain during this condition. Under physiological conditions, the organism will produce ROS and superoxide anion radicals constantly, most of which will be scavenged by the antioxidant enzyme system, including SOD, glutathione peroxidase (GSH-Px), and CAT. However, excessive production of oxygen free radicals together with antioxidant deficiency in the brain causes pathologic changes that are a risk factor for DE [22, 23]. In primary hippocampal neurons, MGO could increase oxidative stress, reduce the activity of antioxidant enzymes, and further induce apoptosis, which explains the possible mechanism of MGO-induced DE [24, 25]. Consistent with these reports, our results also showed that MGO upregulated the level of MDA and downregulated the contents of SOD, CAT, and total GSH, which was markedly reversed by Rb1. These results suggest that the protective effects of Rb1 on MGO-induced SH-SY5Y injury are mediated through inhibiting oxidative stress. In agreement with a former study [26], this experiment proved that MGO increased the generation of ROS, which could dampen the mitochondrial antioxidant defense system and cause oxidative damage [27]. MGO can trigger abnormal opening of the mitochondrial transition pore by the collapse of MMP [28]. As a result, the outer membrane of the mitochondria releases cytochrome C, which recruits its receptor apoptotic protease activating factor 1, ATP, and pro-caspase-9 in the cytoplasm to form an apoptosome. Subsequently, pro-caspase-9 is activated and transformed to caspase-9, and pro-caspase-3 is cleaved to active caspase-3, leading to apoptosis [29]. In contrast, the anti-apoptotic protein Bcl-2, residing in the outer mitochondrial membrane, can form complexes with the pro-apoptosis protein Bax to inhibit the apoptosis induced by mitochondrial damage [30]. Bcl-2 also has a neuroprotective function in the brain [31]. Our results showed that MGO caused the increase in ROS levels and the decrease in MMP, while Rb1 decreased the ROS
generation and alleviated mitochondria dysfunction. From the Hoechst 33342/PI and Annexin V/PI staining, we found MGO caused an elevation of both early stage and later stage apoptosis, and Rb1 treatment significantly reduced the detrimental effects of MGO. Meanwhile, the expression of cleaved caspase-9 and cleaved caspase-3 were increased, and the Bcl-2/Bax ratio was decreased after MGO incubation, and these effects were all suppressed by Rb1. These results suggest that Rb1 could protect SH-SY5Y cells from MGO-induced injury through inhibiting ROS and apoptosis. The PI3K/Akt signaling pathway can regulate the survival, migration, metabolism, and proliferation of cells. Considerable studies have also illustrated that the activation of the PI3K/Akt signaling pathway is involved in anti-apoptosis and antioxidative stress in DE [32, 33]. As a major downstream effector protein of PI3K, Akt directly inhibits pro-apoptosis signals, including Bax [34], to protect neurons. Therefore, we identified the protein expression of Akt and level of phosphorylated Akt (p-Akt). The results showed that MGO inhibited the activation of the PI3K/Akt signaling pathway in SH-SY5Y cells, and Rb1 could reverse this alteration. In addition, inhibition of PI3K/Akt signaling partly abolished the cytoprotective effect of Rb1. Taken together, these results demonstrated that Rb1 enhanced the antioxidative stress and anti-apoptosis capacity of SH-SY5Y cells by activating the PI3K/Akt signaling pathway. 5. Conclusion Taken together, the present study revealed that ginsenoside Rb1 effectively protects SH-SY5Y cells from MGO-induced oxidative stress and apoptosis. The mechanism is probably through the activation of the PI3K/Akt signaling pathway. These findings may provide new avenues of research for the prevention and treatment of DE. Acknowledgements This research was supported by the Major Program of the National Natural Science Foundation of China (NO. 81891012). The authors have declared that no competing interests exist. In addition, Fengwei Nan wants to thank Yunfeng Zhou for his loyalty, his patience, and his love all these years. References [1]
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Highlights Methylglyoxal plays a key role in diabetic encephalopathy, and it exerts a detrimental effect featured by oxidative stress and apoptosis on SH-SY5Y cells. Ginsenoside Rb1 protected SH-SY5Y cells from methylglyoxal-induced oxidative stress and apoptosis. The neuroprotective mechanisms of ginsenoside Rb1 might be involved in regulating the PI3K/Akt signaling pathway.