Salvianolic acid A protects human SH-SY5Y neuroblastoma cells against H2O2-induced injury by increasing stress tolerance ability

doi:10.1016/j.bbrc.2012.04.021

Biochemical and Biophysical Research Communications

Volume 421, Issue 3, 11 May 2012, Pages 479-483

https://doi.org/10.1016/j.bbrc.2012.04.021 Get rights and content

Abstract

Salvianolic acid A (Sal A) is a polyphenol extracted from the root of the Salvia miltiorrhiza bunge. Hydrogen peroxide (H₂O₂) is a major reactive oxygen species (ROS), which has been implicated in stroke and other neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. In this study, we investigated the neuroprotective effects of Sal A in human SH-SY5Y neuroblastoma cells against H₂O₂-induced injury. Our results showed that cells pretreated with Sal A exhibited enhanced neuronal survival and that this protection was associated with an increase in adenosine triphosphate (ATP) and the stabilization of mitochondrial membrane potential. In addition, Sal A markedly decreased the excessive activation AMP-activated protein kinase (AMPK) and the serine–threonine protein kinase, Akt, in SH-SY5Ycells induced by H₂O₂. In conclusion, our results demonstrated that Sal A protects SH-SY5Y cells against H₂O₂-induced oxidative stress and these protective effects are related to stress tolerance and not energy depletion via inhibition of the AMPK and Akt signaling pathway.

Highlights

► Sal A inhibits the activation of AMPK. ► Sal A inhibits the activation of Akt. ► The protective effects of Sal A are related to stress tolerance via inhibition of AMPK and Akt.

Introduction

Salvianolic acid A (Sal A) is a polyphenol extracted from the root of Salvia miltiorrhiza bunge (also known as Danshen), which has been used for treatment of ischemic cardiocerebral vascular diseases thousands of years in China [1]. Growing evidence suggests that Sal A may account for the cardiocerebral protective properties of Danshen [2], [3], [4], [5], [6]. Recently, several molecular mechanisms of Sal A pharmacological activity have been investigated. Currently, several studies have revealed that Sal A plays an important role in a variety of cellular activities, including potent antioxidant activity [7], anti-apoptotic activity [8], improving rCBF [9] and anti-inflammatory effects [10], among others. Sal A has also been shown to function as a natural inhibitor of protein–protein interactions mediated by Src-family SH2 domains [11]. Moreover, Sal A can inhibit platelet activation and arterial thrombosis via inhibition of phosphoinositide 3-kinase [12]. Furthermore, Sal A suppresses the lipopolysaccharide-induced NF-kappaB signaling pathway by targeting IKKbeta [10]. Although our understanding of the role of Sal A in several diseases has grown over the past few years, further insight into the effects and mechanisms of Sal A is needed to utilize Danshen’s therapeutic potential to treat ischemic cardiocerebral vascular diseases.

Reactive oxygen species (ROS) have been hypothesized to play an important role in the coordinated cellular signaling [13]. The H₂O₂ ROS plays a crucial role during neuronal cell death in stroke and other neurodegenerative conditions [7], [14]. Human SH-SY5Y neuroblastoma cells are widely used as an experimental cell system for the study of neuronal cell damage [14], [15]. In this study, we evaluated H₂O₂-induced damage to human SH-SY5Y neuroblastoma cells to examine the protective effects of Sal A.

The AMP-activated protein kinase (AMPK), a serine threonine kinase, is a key metabolic and stress sensor/effector [16]. Growing evidence indicates that the serine–threonine protein kinase, Akt (also referred to as protein kinase B, PKB), is an important mediator of cell survival. Several reports indicate that Akt plays a key role in a broad spectrum of vital cellular functions [17], [18]; thus, it is important to analyze the role of Akt in a given model and environment. However, the effects of Sal A on AMPK and Akt levels in neurocyte damage have not yet been reported.

Sal A possesses one of the most important cardiocerebral protective properties of Danshen. We investigated the neuroprotective effects mechanisms of Sal A, with regard to cell viability, ATP content, mitochondrial membrane potential and the expression of AMPK and Akt during H₂O₂-induced injury in SH-SY5Y cells.

Section snippets

Cell culture and treatments

Human SH-SY5Y neuroblastoma cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) and F-12 (GIBCO, Gaithersburg) supplemented with 10% fetal bovine serum (FBS, Hyclone) in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C. The media was replaced every 2 days. Prior to the experiments, the SH-SY5Y cells were plated in 96-well plates at a density of approximately 1.5 × 10⁴ cells per well (for MTT, ATP and mitochondrial membrane potential assays) and in 6-well plates at 8 × 10⁶ cells

The effects of Sal A on the viability of SH-SY5Y cells exposed to H₂O₂

To determine the optimal concentration of H₂O₂, SH-SY5Y cells were cultured with varying concentrations of H₂O₂ for 24 h. Because our results showed a decreased level of the OD value (by 71%) after treatment with 200 μM of H₂O₂ (Fig. 1A), we selected a 24-h treatment of 200 μM H₂O₂ for subsequent experiments in the present study. As shown in Fig. 1B, the viability of the SH-SY5Y cells exposed to 200 μM H₂O₂ for 24 h was reduced compared with the normal group. In addition, co-treatment with Sal A

Discussion

In this study, we investigated the effects of Sal A on the injury of SH-SY5Y cells induced by H₂O₂. The decreasing survival rates of SH-SY5Y cells induced by H₂O₂ were increased after treatment with Sal A, and this protection was associated with the increased production of ATP and stabilization of the mitochondrial membrane potential. These findings indicated a protective effect of Sal A on H₂O₂-induced injury of SH-SY5Y cells, which is consistent with previous studies [8], [19].

Based on

References (36)

J.Y. Han et al.
Ameliorating effects of compounds derived from Salvia miltiorrhiza root extract on microcirculatory disturbance and target organ injury by ischemia and reperfusion
Pharmacol. Ther.
(2008)
T.J. Lin et al.
Protection by salvianolic acid A against adriamycin toxicity on rat heart mitochondria
Free Radic. Biol. Med.
(1992)
S.B. Wang et al.
Du GH, Cardioprotective effect of salvianolic acid A on isoproterenol-induced myocardial infarction in rats
Eur. J. Pharmacol.
(2009)
X.J. Wang et al.
Salvianic acid A protects human neuroblastoma SH-SY5Y cells against MPP+-induced cytotoxicity
Neurosci. Res.
(2005)
M.K. Tang et al.
Du GH
Effect of salvianolic acids from Radix Salviae miltiorrhizae on regional cerebral blood flow and platelet aggregation in rats, Phytomedicine.
(2002)
B. Sperl et al.
Natural product inhibitors of protein–protein interactions mediated by Src-family SH2 domains
Bioorg. Med. Chem. Lett.
(2009)
Z.S. Huang et al.
Salvianolic acid A inhibits platelet activation and arterial thrombosis via inhibition of phosphoinositide 3-kinase
J. Thromb. Haemost.
(2010)
S.R. Heo et al.
P62 protects SH-SY5Y neuroblastoma cells against H₂O₂-induced injury through the PDK1/Akt pathway
Neurosci. Lett.
(2009)
M.P. Scheid et al.
Unravelling the activation mechanisms of protein kinase B/Akt
FEBS Lett.
(2003)
X.J. Wang et al.
Effect of salvianolic acid A on lipid peroxidation and membrane permeability inmitochondria
J. Ethnopharmacol.
(2005)

S. Janelidze et al.

Alterations of Akt1 (PKBalpha) and p70(S6K) in transient focal ischemia

Neurobiol. Dis.

(2001)

M. Shibata et al.

Upregulation of Akt phosphorylation at the early stage of middle cerebral artery occlusion in mice

Brain Res.

(2002)

J. Ruffels et al.

Activation of ERK1/2, JNK and PKB by hydrogen peroxide in human SH-SY5Y neuroblastoma cells: role of ERK1/2 in H₂O₂-induced cell death

Eur. J. Pharmacol.

(2004)

L.D. McCullough et al.

Pharmacological inhibition of AMP-activated protein kinase provides neuroprotection in stroke

J. Biol. Chem.

(2005)

J.C. Dong et al.

Beneficial effects of acetylsalvianolic acid A on focal cerebral ischemic rats subjected to middle cerebral artery thrombosis

Yao Xue Xue Bao.

(1996)

G.H. Du et al.

Protective effects of salvianolic acid A against impairment of memory induced by cerebral ischemia-reperfusion in mice

Chin. Med. J.

(1997)

T. Wang et al.

Salvianolic acid A exerts antiamnesic effect on diazepam-induced anterograde amnesia in mice

Neurochem. Res.

(2011)

G.H. Du et al.

Protective effects of salvianolic acid A against impairment of memory induced by cerebral ischemia-reperfusion in mice

Yao Xue Xue Bao.

(1995)

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Salvianolic acids are the most water-soluble compounds in S. miltiorrhiza, of which salvianolic acid A (Sal A) and salvianolic acid B (Sal B) are most abundantly found. Sal A possesses anti-oxidative (Du & Zhang, 1995), anti-apoptotic (Wang & Xu, 2005), and anti-inflammatory (Oh, Oh, Mun, Seo, & Lee, 2011) properties, and is reported to protect human SH-SY5Y neuroblastoma cells against H2O2-induced injury by increasing the stress tolerance ability (Zhang et al., 2012). Sal B has also been studied extensively, and is shown to have a wide range of pharmacological effects including anti-oxidation, apoptosis inhibition, coronary artery dilation, and aggregation platelet prevention (Wang et al., 2013; Ren et al., 2016).
Salvianolic acid B (Sal B), a natural compound extracted from Salvia miltiorrhiza (Danshen), is well known for its anti-oxidative activity and treatment of cardiovascular diseases. However, little is known about the functional roles of Sal B in white adipocytes. Therefore, this study aimed to investigate the functions of Sal B in 3T3-L1 white adipocytes, with a focus on white fat browning effect. Exposure to Sal B significantly increased the expressions of beige-specific genes and thermogenic marker proteins. Sal B also suppressed lipogenesis and enhanced lipolysis in white adipocytes. A mechanistic study revealed that Sal B stimulates white fat browning via β3-AR and ERK signaling pathways in an independent manner. In conclusion, our findings indicate that Sal B is beneficial in the prevention and treatment of obesity, at least in part by promoting thermogenesis.
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The neuroprotective mechanisms could be attributed to inhibiting inflammatory responses, nitrotyrosine formation, and NF-κB signaling induction [170]. Sal A treatment also showed to attenuate eNOS uncoupling and inhibited ONOO− production in the ischemic brains in vivo, and prevented H₂O₂-mediated injury in cultured human SH-SY5Y neuroblastoma cells in vitro [173,174]. Sal A also inhibited the adhesion molecule ICAM-1 in endothelial cells and decreased the adhesion of granulocytes in vitro [175].
Oxidative/nitrosative stress and neuroinflammation are critical pathological processes in cerebral ischemia-reperfusion injury, and their intimate interactions mediate neuronal damage, blood-brain barrier (BBB) damage and hemorrhagic transformation (HT) during ischemic stroke. We review current progress towards understanding the interactions of oxidative/nitrosative stress and inflammatory responses in ischemic brain injury. The interactions between reactive oxygen species (ROS)/reactive nitrogen species (RNS) and innate immune receptors such as TLR2/4, NOD-like receptor, RAGE, and scavenger receptors are crucial pathological mechanisms that amplify brain damage during cerebral ischemic injury. Furthermore, we review the current progress of omics and systematic biology approaches for studying complex network regulations related to oxidative/nitrosative stress and inflammation in the pathology of ischemic stroke. Targeting oxidative/nitrosative stress and neuroinflammation could be a promising therapeutic strategy for ischemic stroke treatment. We then review recent advances in discovering compounds from medicinal herbs with the bioactivities of simultaneously regulating oxidative/nitrosative stress and pro-inflammatory molecules for minimizing ischemic brain injury. These compounds include sesamin, baicalin, salvianolic acid A, 6-paradol, silymarin, apocynin, 3H-1,2-Dithiole-3-thione, (−)-epicatechin, rutin, Dl-3-N-butylphthalide, and naringin. We finally summarize recent developments of the omics and systematic biology approaches for exploring the molecular mechanisms and active compounds of Traditional Chinese Medicine (TCM) formulae with the properties of antioxidant and anti-inflammation for neuroprotection. The comprehensive omics and systematic biology approaches provide powerful tools for exploring therapeutic principles of TCM formulae and developing precision medicine for stroke treatment.
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A new manganese(III) complex [MnL¹(DCA)(H₂O)](H₂O)_, 1 [H₂L¹ is the chelating ligand N,N′-bis(2-hydroxy-3-methoxybenzylidene)-1,2-diaminopropane, and DCA is dicyanamide], has been prepared and characterized by different analytical and spectroscopic techniques. The tetragonally elongated octahedral geometry for the manganese coordination sphere was revealed by X-ray diffraction studies for 1. The antioxidant behavior of this complex and other manganese(III)-salen type complexes was tested through superoxide dismutase and catalase probes, and through the study of their neuroprotective effects in SH-SY5Y neuroblastoma cells. In this human neuronal model, these model complexes were found to improve cell survival in an oxidative stress model. During studies aimed to getting a better understanding of the kinetics of the processes involved in this antioxidant behavior, an important effect on the solvent in the kinetics of reaction of the complexes with H₂O₂ was revealed that suggests a change in the mechanism of reaction of the complexes. The kinetic data in methanol and buffered aqueous solutions correlate well with the results of the test of catalase activity, thus showing that the rate determining step in the catalytic cycle corresponds to the initial reaction of the complexes with H₂O₂.
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Sal B was proved to be a potential candidate for treatment of Alzheimeŕs disease [6], liver fibrosis [7], and traumatic brain injury [8]. Sal A was reported to have antioxidant [9], radical scavenging [10], neuroprotection [11], cardiovascular and cerebrovascular protection effects [12–14]. And in some respects, Sal A is more active than Sal B [15].
Salvianolic acid A is the major bioactive compound in Danshen, however, due to the chemical instability and low content in Danshen, it is difficult to extract amount of salvianolic acid A. Therefore, this study was to establish an effective strategy for obtaining adequate amount of salvianolic acid A, subcritical water extraction was used to degrade salvianolic acid B and prepare salvianolic acid A. Different reaction conditions including temperature, time, concentration and pH value in subcritical water were investigated. Under 40 mg/mL of reactant concentration, 180 °C of temperature, 4.0 of pH value and 60 min of reaction time, the highest yield rate of salvianolic acid A reached 34.86%. Then, the degradation products were successfully separated by pH-zone-refining counter-current chromatography with the solvent system Pet–EtAc–n-BuOH–H₂O (2:3:1:9, v/v), where 10 mM TFA was added in stationary phase and 10 mM NH₃·H₂O in mobile phase. As a result, a total of 227.3 mg of salvianolic acid A at 98.2% purity, 38.9 mg of danshensu at 99.3% purity, 9.5 mg of salvianolic acid D at 92.7% purity, and 32.8 mg of protocatechuic aldehyde at 93.1% purity were obtained from 1.2 g degradation products of salvianolic acid B by one-step purification. The results demonstrated that the combinative application of subcritical water and pH-zone-refining counter-current chromatography is a potential technique for the preparative separation of salvianolic acid A from salvianolic acid B.
Effects of the Nrf2 protein modulator salvianolic acid a alone or combined with metformin on diabetes-associated macrovascular and renal injury
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Nuclear factor E2-related factor 2 (Nrf2) is considered a promising target against diabetic complications such as cardiovascular diseases and diabetic nephropathy. Herein, we investigated the effects of a potential Nrf2 modulator, salvianolic acid A (SAA), which is a natural polyphenol, on diabetes-associated macrovascular and renal injuries in streptozotocin-induced diabetic mice. Given that lowering glucose is the first objective of diabetic patients, we also examined the effects of SAA combined with metformin (MET) on both complications. Our results showed that SAA significantly increased the macrovascular relaxation response to acetylcholine and sodium nitroprusside in diabetic mice. Interestingly, treatment with SAA alone only provided minor protection against renal injury, as reflected by minor improvements in impaired renal function and structure, despite significantly reduced oxidative stress observed in the diabetic kidney. We demonstrated that decreased oxidative stress and NF-κB p65 expression were associated with SAA-induced expression of Nrf2-responsive antioxidant enzymes heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase (quinone) 1 (NQO-1), and glutathione peroxidase-1 (GPx-1) in vivo or in vitro, which suggested that SAA was a potential Nrf2 modulator. More significantly, compared with treatment with either SAA or MET alone, we found that their combination provided further protection against the macrovascular and renal injury, which was at least partly due to therapeutic activation of both MET-mediated AMP-activated protein kinase and SAA-mediated Nrf2/antioxidant-response element pathways. These findings suggested that polyphenol Nrf2 modulators, especially combined with drugs activating AMP-activated protein kinase, including hypoglycemic drugs, are worthy of further investigation to combat diabetic complications.

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¹: Address: Qingdao Municipal Hospital, Qingdao 266011, PR China.

²: Address: Tianjin Institute of Pharmaceutical Research, 308 Anshan Xidao, Nankai District, Tianjin 300193, PR China.

³: Address: Center for Critical Illness Research, Lawson Health Research Institute, Department of Medicine and Pathology, University of Western Ontario, London, Canada.

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Salvianolic acid A protects human SH-SY5Y neuroblastoma cells against H2O2-induced injury by increasing stress tolerance ability

Abstract

Highlights

Introduction

Section snippets

Cell culture and treatments

The effects of Sal A on the viability of SH-SY5Y cells exposed to H2O2

Discussion

Pharmacol. Ther.

Free Radic. Biol. Med.

Eur. J. Pharmacol.

Neurosci. Res.

Effect of salvianolic acids from Radix Salviae miltiorrhizae on regional cerebral blood flow and platelet aggregation in rats, Phytomedicine.

Bioorg. Med. Chem. Lett.

J. Thromb. Haemost.

Neurosci. Lett.

FEBS Lett.

J. Ethnopharmacol.

Neurobiol. Dis.

Brain Res.

Eur. J. Pharmacol.

J. Biol. Chem.

Beneficial effects of acetylsalvianolic acid A on focal cerebral ischemic rats subjected to middle cerebral artery thrombosis

Yao Xue Xue Bao.

Protective effects of salvianolic acid A against impairment of memory induced by cerebral ischemia-reperfusion in mice

Chin. Med. J.

Salvianolic acid A exerts antiamnesic effect on diazepam-induced anterograde amnesia in mice

Neurochem. Res.

Protective effects of salvianolic acid A against impairment of memory induced by cerebral ischemia-reperfusion in mice

Yao Xue Xue Bao.

Salvianolic acid A protects human SH-SY5Y neuroblastoma cells against H₂O₂-induced injury by increasing stress tolerance ability

The effects of Sal A on the viability of SH-SY5Y cells exposed to H₂O₂