Introduction
Glutamate excitotoxicity is a major factor in ischemia-induced neuronal death (Nishizawa, 2001). Excess release of glutamate from presynaptic membranes induced by ischemia overactivates glutamate receptors leading to a series of events including intracellular calcium overload, excessive ROS production and mitochondrial stress and finally neuronal death (Benveniste et al., 1984; Lipton and Rosenberg, 1994). However, based on accumulating evidence in the literature, all clinical trials using glutamate receptor inhibitors have failed (Koh and Choi, 1991; Morris et al., 1999; Albers et al., 2001; Ikonomidou and Turski, 2002) although some of the inhibitors reduced ischemic damage in animal experiments (Lin et al., 1993; Reyes et al., 1998; Cai, 2006). It is known that glutamate receptors play important roles in maintaining physiological functions such as excitatory signal transduction, learning and memory (Mayer and Westbrook, 1987; Newcomer et al., 2000). Therefore, a more promising strategy for treating ischemic stroke may be through selectively blocking excitotoxicity while preserving important physiological aspects of glutamate receptor subunit function (Cho et al., 2010).
Previous studies have identified three NMDAR subunits: NR1, NR2 (A–D) and NR3 (A,B). Functional NMDARs are heterotetramers composed of two glycine–biding NR1 subunits and two glutamate-binding NR2 subunits, whereas NR1/NR3 heterotetramers can be combined by glycine (Chatterton et al., 2002; Mayer and Armstrong, 2004; Paoletti, 2011). Different NR2-containing receptors (NR1/NR2A, or NR1/NR2B heterotetramers) exhibit different biophysical and pharmacological properties (Cull-Candy and Leszkiewicz, 2004; Furukawa et al., 2005; Chen and Wyllie, 2006). NR2A subunits are primarily located at intrasynaptic sites, whereas NR2B subunits are predominantly located at extrasynaptic sites (Stocca and Vicini, 1998; Rumbaugh and Vicini, 1999; Tovar and Westbrook, 1999; Traynelis et al., 2010). Functionally, NR2A subunits play a neuroprotective role by activating cellular CREB or Akt pathways (Hardingham and Bading, 2010; Luo et al., 2011; Lai et al., 2014). Activation of NR2A will induce phosphorylation of CREB which is associated with BDNF expression and contributes to neuronal survival (Chen et al., 2008). It has been reported that some drugs may be neuroprotective against ischemia via enhancing CREB activity (Raval et al., 2009; Zhang et al., 2010). Conversely, activation of the extrasynaptic NR2B subunit will trigger apoptotic pathways by increasing ROS levels and prohibiting CREB expression (Léveillé et al., 2008; Hardingham and Bading, 2010; Gladding and Raymond, 2011). When animals experience ischemic insult, nNOS will translocate to the cell membrane to form the NR2B-PSD95-nNOS complex that activates nNOS to produce more NO and causes severe neuronal injury. Some agents have recently been tested in the rat MCAO model and stroke primates showing that disruption of nNOS-PSD95 or NR2B-PSD95 interaction reduced infarct area in ischemic models (Zhou et al., 2010; Cook et al., 2012). Whether both NR2A and NR2B subunits could be simultaneously regulated to achieve neuroprotection by pharmacological drugs remains unknown.
Lycium barbarum (Gouqi or wolfberry) is well known as a traditional Chinese medicine and healthy food supplement in China and other countries (Amagase et al., 2009). Lycium barbarum polysaccharide (LBP) is a mixed compound extracted from the fruits of Guoqi, composing primarily of rhamnose, arabinose, xylose, galactose, mannose and galacturonic acid (Zou et al., 2010). Recent years, many studies have investigated LBP’s structures, bioactive components and degradation. Studies have found that the water-soluble LBP containing 3.75% proteins has a β-D-(1 → 6)-galactan as a backbone with highly branched polysaccharide (Wang Z. et al., 2014; Yuan et al., 2016). A new study reported that a p-LBP was isolated and purified from LBP-and p-LBP was a homogeneous heteropolysaccharide as a pectin molecule with an average molecular weight of 64 kDa, approximately 87 nm hollow sphere in 0.05 mol/L sodium sulfate solution (Liu et al., 2016). Many studies have shown that LBP has strong anti-oxidative effects in diverse injury types, including doxorubicin-induced cardiotoxicity (Xin et al., 2011) and oxidative liver injury (Wu et al., 2010; Xiao et al., 2012). In addition, some studies showed that LBP plays beneficial roles in aging (Tang and He, 2013), hypoglycemia (Zhu et al., 2013) and anti-radiation or chemotherapy damages (Gong et al., 2005). Concerning nervous system damage, LBP protected ganglion cells against acute ocular hypertension (Mi et al., 2012), partial optic transection injuries (Li et al., 2013) and retinal ischemia/reperfusion injury (Li et al., 2011). Furthermore, LBP can prevent cortical neurons from damage against ischemic insults through anti-oxidative and anti-apoptotic mechanisms (Rui et al., 2012; Wang T. et al., 2014).
LBP appears to have broad benefits in a variety of injury types by regulating different pathways. Based on the idea that different NMDA receptor subunits have distinct functions, we hypothesized that LBP exhibits its neuroprotective effect against ischemic injury by regulating NR2A and NR2B signaling pathways. The present study investigated the underlying mechanisms of LBP-induced neuroprotection in in vivo ischemia and in vitro oxygen-glucose deprivation (OGD) models and unraveled that indeed both NR2A and NR2B receptor signaling pathways play crucial role in the actions of LBP.