Stroke remains a leading cause of death and adult disability worldwide. Ischemic stroke accounts for approximately 80% of all strokes (Tsuchiya et al. 1992; Feigin et al. 2003). Ischemic insult produces excessive free radicals which are neurotoxic by inducing apoptotic cell death in neurons. The neurotoxicity mediated by free radicals was supported by reduced ischemic injury following application of antioxidant enzymes and scavengers (Naritomi 2001). Thus, it is recognized that free radical scavengers can act as potential neuroprotective agents.
Scutellaria baicalensis Georgi (Huangqin) has been widely used as antibacterial and anti-inflammatory agents for many centuries in the traditional Chinese herbal medicine. Baicalein (Bai) is the most effective antioxidant among the major flavonoids isolated from the roots of Scutellaria baicalensis. In this regard, it has been reported to scavenge reactive oxygen species (ROS), including superoxide , H2O2, and hydroxyl radicals (Hamada et al. 1993; Hanasaki et al. 1994). Baicalein also has the ability to strongly inhibit iron-dependent lipid peroxidation in microsomes (Gao et al. 1995) and mitochondria (Miyahara et al. 1993; He et al. 2009). Results from our laboratory and others have demonstrated that baicalein has the ability to reduce oxidative damage and exert potent neuroprotective effect in a variety of cell types and animal models including experimental ischemia (van et al. 2006; Lapchak et al. 2007; Liu et al. 2007; Jin et al. 2008), indicating that Bai could be an effective pharmacotherapy for the prevention or treatment of neurodegenerative diseases such as stroke. However, the underlying mechanism has not been well elucidated.
Reactive oxygen species derived from ischemia/reperfusion are associated with phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway that leads to neuronal survival or death (Taylor and Crack 2004; Crack and Taylor 2005). PI3K/AKT is a major cell survival pathway that has been extensively studied. PI3K/Akt pathway promotes cellular survival by phosphorylating and inhibiting death-inducing proteins, including GSK3, Bcl-2/Bcl-xL-associated death protein (BAD), caspase 9, and forkhead transcription factor like 1 (Datta et al. 1997; Cardone et al. 1998; Brunet et al. 1999; Chan 2004; Woodgett 2005). The activity of AKT depends on the availability of phosphoinisitidylinositol-3,4,5-triphosphate (PIP3), which is generated by the enzyme PI3K. PTEN, the phosphatase and tensin homolog deleted on chromosome 10, was originally identified as a tumor suppressor gene mutated in a large percentage of human cancers. PTEN has a critical role in antagonizing PI3K pathways by dephosphorylates PIP3 and converts it back to PIP2. Thus, PTEN is considered to be a key negative regulator of the PI3K/Akt pathway (Waite and Eng 2002). Considering the key role of PI3K/Akt and PTEN in cell survival in models of neurotoxicity, we sought to determine whether PI3K/Akt and PTEN is involved in the neuroprotective effect of Bai. Two stroke models, one of temporary ischemia with reperfusion at 2 h after stroke onset, and one of permanent ischemia was used to assess whether these effects were dependent on the type of ischemia. In addition, oxygen and glucose deprivation (OGD) in primary cultured neurons was used to mimic ischemic insult in vitro. We have demonstrated that baicalein, a known antioxidant, reduce oxidative stress generated by ischemia/reperfusion and promote cell survival involving PI3K/Akt and PTEN signal pathway.
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- Material and methods
The brain is particularly susceptible to oxidative stress because it consumes a large quantity of oxygen. Although oxygen is necessary for life, it paradoxically produces ROS as a by-product of its metabolism. Under normal conditions, the rate of free radicals formation is equal to that of their elimination. However, during ischemia and reperfusion, this balance is perturbed either because of increased free radicals production or decreased activity of cellular defense systems (Valko et al. 2007), which lead to free radicals massively accumulate in the brain. Those radicals are redox active and have the potential to react with and damage nearby cellular targets including lipids, proteins and DNA, which is followed by more delayed post-ischemic inflammation and apoptosis, and these events are involved in the progression and expansion of brain injury (Peters et al. 1998; Hata et al. 2000). Thus, oxidative stress are believed to contribute to neuronal loss after ischemia/reperfusion (Andersen 2004; Margaill et al. 2005) and the scavenger of free radicals is considered to be important for achieving neuroprotection against ischemia/reperfusion injury.
Arachidonic acid metabolism is a potential source for ROS generation during ischemia and reperfusion. As a result of the activation of phospholipases in responding to brain ischemia, increased arachidonic acid release occurs (Katsuki and Okuda 1995) and is metabolized by three enzyme systems: cyclooxygenase, LOX, and epoxygenase. Metabolism of arachidonic acid by these enzymes produce free radicals and peroxides (Siesjo and Katsura 1992; Paller and Jacob 1994; Phillis et al. 2006). Lipoxygenase (LOX), one of the major metabolic enzymes, catalyzes the incorporation of molecular oxygen into specific positions of arachidonic acid. Based on the position of oxygen insertion, LOX is classified as 5-, 12-, or 15-LOX (Shimizu and Wolfe 1990). 12-LOX is the major LOX found in the brain. Blockage 12/15-LOX activation prevents ROS generation and accumulation during cerebral ischemia. The natural product Bai has been identified as a specific inhibitor of 12/15-LOX. As a polyphenol which belongs to the flavone subgroup, Bai also has superior free radical scavengering and antioxidant effects because of its o-trihydroxyl structure in the A ring (Gao et al. 1999) and lipophilic character (Saija et al. 1995). Gao et al. reported Bai scavenges hydroxyl radical, DPPH (1,1-Diphenyl-2-Picrylhydrazyl) radical, and alkyl radical in a dose-dependent manner in vitro, inhibits lipid peroxidation of rat brain cortex mitochondria induced by Fe2•−ascorbic acid, 2,2′-azobis (2-amidino-propane) dihydrochloride (AAPH), or NADPH, protects human neuroblastoma SH-SY5Y cells against H2O2-induced injury (Gao et al. 1999). Our previous studies showed that Bai inhibited ischemic neuronal cell loss related to its antioxidant action (Liu et al. 2007). In this study, a rapid but transient increase in the intracellular ROS level was observed shortly after OGD. As we know, the major oxidative stress produced by superoxide is derived from its reaction with nitric oxide yielding peroxynitrite (ONOO−), a highly reactive species and much more toxic than , to elicit nitrosative damage. A characteristic reaction of ONOO− is the nitration of protein-bound tyrosine residues. Neurons are capable of generating ONOO− because of their capacity to simultaneously produce and NO. Our results also demonstrated increment of 3-nitrotyrosine (3-NT) formation produced by OGD/R treatment. Excessive ROS and 3-NT plays an important role in induction of apoptotic death in cultured cortical neurons. When Bai was applied to neurons exposure to OGD/R, ROS, and 3-NT formation was markedly decreased accompanied by increased cell survival. This study was reported for the first time that Bai protects cortical neuronal cells from OGD, the most often used in vitro model mimicking metabolic aspects of ischemia, induced cell death by inhibiting apoptosis, which greatly extends previous reports on the effects of Bai on systemic tissues. In agreement with this, our investigation in vivo demonstrated that Bai administration significantly reduced infarct size and apoptosis induced by transient MCAO and reperfusion in rats, which is consistent with the previous reports (onso-Galicia et al. 1999; van et al. 2006). In contrast to permanent MCAO, reperfusion prompts a short-lasting steep maximum to nearly fivefold increase of ROS concentrations, while permanent vessel occlusion led to a more gradual increase by about twofold levels at 3-h post-occlusion (Peters et al. 1998). Deleterious effect of superoxide radicals is enhanced by reperfusion and is a leading cause in reperfusion injury. Thus, suppression ROS plays a more predominant role in rescuing cells from reperfusion injury. However, the restoration of flow makes the therapeutic agent to reach the ischemic tissue to minimize infarct development. These reasons may explain the better outcome for Bai administration in transient MCAO compared with permanent MCAO.
PI3K/Akt regulates the survival response against oxidative stress-associated neuronal apoptosis (Dudek et al. 1997; Hong et al. 2001; Manning and Cantley 2007). In the CNS, decreased Akt activity has been linked to the neuronal death induced by ischemia or hypoxia (Hirai et al. 2003, 2004; Luo et al. 2003). In contrast, increased Akt activity contributes to the neuroprotection induced by hypothermia (Zhao et al. 2005) or hypoxic preconditioning (Zhang et al. 2007; Wang et al. 2008). Constitutive activation of Akt signaling is sufficient to block cell death induced by a variety of apoptotic stimuli. Phosphorylation of Akt (Ser473) is required for Akt activation. Several downstream targets of Akt have been recognized as apoptosis-regulatory molecules. Activated Akt promotes cell survival and suppresses apoptosis by phosphorylation and inhibition of several downstream substrates, including GSK3β (Cross et al. 1995), a mechanism by which neurons are proposed to become resistant to apoptotic stimuli (Hetman et al. 2000). BAD is also the target of Akt. As a proapoptotic member of the Bcl-2 family, BAD promote cell death by binding and neutralizing the function of anti-apoptotic Bcl-2. Phosphorylation of BAD promotes binding to 14-3-3 proteins to be sequestered in the cytosol and to prevent an association between BAD with Bcl-2 (Yang et al. 1995; Zha et al. 1996; Datta et al. 2000). Akt inhibit the apoptotic activity of BAD by phosphorylation of BAD at Ser136 (Datta et al. 1999). Following cell stress, the loss of Akt activity leads to BAD dephosphorylation and translocation to mitochondria, where it binds with Bcl-2 and activates the mitochondrial cell death pathway to release cytochrome c into cytosol. In this study, we demonstrated that Bai treatment enhanced the reduced Akt phosphorylation after OGD/R. Pharmacologic inhibition PI3K or silencing Akt expression by siRNA impaired the ability of Bai to protect against OGD/R-induced cortical neurons death. Moreover, Bai increased phosphorylation of GSK3βand BAD, and inhibited OGD/R-induced loss of Bcl-2 from mitochondria. Cytochrome c release in cytosol was sequentially blocked. These are consistent with the results that baicalein inhibit caspase 3 activation and apoptotic death in transient MCAO rats. In addition, pre-treatment with LY294002, the specific inhibitor of PI3K, blocked baicalein to increase Akt phosphorylation and the following targets when exposure to OGD/R. We also found the decreased intracellular ROS level by baicalein after OGD/R was abolished in the presence of LY294002, further verified that PI3K/Akt-mediated neuroprotection regulated oxidative stress response (Di et al. 2006).
The biological effects of Akt are determined by the balance between the activity of PI3K and PTEN. PTEN is a major negative regulator of the PI3K/Akt signaling pathway. The phosphorylation of three specific residues Ser380/Thr382/383 of PTEN is required for its biological activity (Torres and Pulido 2001). Unlike AKT, PTEN phosphorylation results in its inactivation, not activation. Dephosphorylation of PTEN increases PTEN activity and reduces PIP3 availability leading to dephosphorylation of AKT. PTEN has been demonstrated to act as an important mediator of ROS production and of mitochondria-dependent apoptosis (Zhu et al. 2006). Over-expression of PTEN increased the sensitivity of hippocampal neurons to excitotoxicity (Gary and Mattson 2002), whereas knockdown PTEN or pharmacological down-regulation of PTEN phosphorylation protected brain tissue from ischemic damage (Ning et al. 2004; Hong et al. 2006). So, we further examined the possibility that PTEN is involved in the effect of Bai on OGD/R-treated cortical neurons. Consistent with this prediction, we found that PTEN is rapidly dephosphorylated after OGD and this dephosphorylation is reversed by Bai. Therefore, it is likely that Bai scavenge free radicals and suppress oxidant stress in ischemia/reperfusion, which causes PTEN to lose its activity and, in turn, increase AKT activity and inhibit the downstream mediated apoptotic cell death.
To summarize, we have demonstrated that baicalein administration have neuroprotective effect against ischemic brain injury both in vivo and in vitro which is related to its ability to scavenge free radicals and mediated by PI3K/Akt and PTEN pathway. The findings provide further insight into the mechanisms through which Bai exerts its beneficial effect on the injured brain and suggests Bai may be a promising agent for clinic therapy of ischemic brain damage.