We have found the Cordyceps sinensis extracts (CSE) significantly reduced focal cerebral ischemic/reperfusion injury. Cerebral ischemic injury can induce the pathological pathway of DAPK1-NMDAR and cause inflammatory reaction, which will lead to the death of never cells. However, researches showed that phytoestrogens has inhibitory effect on this injury. GPR30 played fast non genomic effects through combining with phytoestrogens and mediating the second messenger, further inhibited the phosphorylation of DAPK1. As a result, we hypothesize that it is isoflavone in Cordyceps sinensis (CSIF) which has estrogenic activity plays a vital role in the treatment of ischemic brain injury. It can inhibit the pathological pathway of DAPK1-NMDAR through combining with GPR30. Then, never cells will be protected from dying and ischemic brain injury will be cured. All of these are our hypothesis.

 
Keywords: Ischemic Brain Injury; Isoflavone in Cordyceps Sinensis (CSIF); G-Protein-Coupled Receptor 30 (GPR30); the Pathological Pathway of DAPK1-NMDAR.  

The cerebral vascular disease (CVD) is one of the common diseases in the elderly, with characteristics of high incidence, high rate of relapse and high morbidity. The disease is given priority to with stroke, especially ischemic stroke. Stroke is the fourth leading cause of death throughout the world [1], and in most causes it is caused by cerebral artery occlusion (ischemic stroke). The subsequent ischemic brain injury is the result of interactions among complex pathophysiological processes such as excitotoxicity, calcium dysregulation, oxidative stress, inflammation, and apoptosis [2]. The current treatment of ischemic stroke in the national and world doesn’t have a unified standard completely [3]. Therefore, studying the ischemic stroke’s molecular mechanism and exploring the prevention measures have a significant social and economic significance in order to reduce the incidence of the disease.

 
The genus Cordyceps is an important kind of medicinal fungi belonging to the Ascomycota, Pyrenomycetes, Hypocreales, Clavicipitaceae [4-8]. The genus Cordyceps are specific macrofungi because of their characteristic parasitic habit on larvae and pupae of insects. Cordyceps, a pleomorphism fungi distributed worldwide, is particularly abundant in tropical forests and humid temperate. Within the genus Cordyceps, over 400 species have been described so far [7,8], of which Cordyceps sinensis, also called as “Winter Worm, Summer Grass”, is recognized as the most famous tonic herb in traditional Chinese medicine (TCM) for centuries. Cordyceps is an abundant resource in nature with various biological activities, and has been used extensively as a tonic and health supplement for subhealth patients, especially seniors, in China and other Asian countries. Till now, numerous bioactive constituents have been extracted, such as cordycepin, isoflavone, polysaccharides, ergosterol, mannitol, adenosine [9,10]. Meanwhile, various pharmacological actions of these chemical constituents have been reported, including antitumour effect, hepatoprotective, inflammatory effects, antioxidant, nephroprotective, anti-apoptotic properties and so on [11-17]. Previous studies found that Cordyceps sinensis extract (CSE) had obvious protective effect on cerebral ischemic rats caused by middle cerebral artery occlusion (MCAO) [18], the specific effective parts and its treatment mechanism are unclear however.

The cerebral vascular disease (CVD) is one of the common diseases in the elderly, with characteristics of high incidence, high rate of relapse and high morbidity. The disease is given priority to with stroke, especially ischemic stroke. Stroke is the fourth leading cause of death throughout the world [1], and in most causes it is caused by cerebral artery occlusion (ischemic stroke). The subsequent ischemic brain injury is the result of interactions among complex pathophysiological processes such as excitotoxicity, calcium dysregulation, oxidative stress, inflammation, and apoptosis [2]. The current treatment of ischemic stroke in the national and world doesn’t have a unified standard completely [3]. Therefore, studying the ischemic stroke’s molecular mechanism and exploring the prevention measures have a significant social and economic significance in order to reduce the incidence of the disease.

 
The genus Cordyceps is an important kind of medicinal fungi belonging to the Ascomycota, Pyrenomycetes, Hypocreales, Clavicipitaceae [4-8]. The genus Cordyceps are specific macrofungi because of their characteristic parasitic habit on larvae and pupae of insects. Cordyceps, a pleomorphism fungi distributed worldwide, is particularly abundant in tropical forests and humid temperate. Within the genus Cordyceps, over 400 species have been described so far [7,8], of which Cordyceps sinensis, also called as “Winter Worm, Summer Grass”, is recognized as the most famous tonic herb in traditional Chinese medicine (TCM) for centuries. Cordyceps is an abundant resource in nature with various biological activities, and has been used extensively as a tonic and health supplement for subhealth patients, especially seniors, in China and other Asian countries. Till now, numerous bioactive constituents have been extracted, such as cordycepin, isoflavone, polysaccharides, ergosterol, mannitol, adenosine [9,10]. Meanwhile, various pharmacological actions of these chemical constituents have been reported, including antitumour effect, hepatoprotective, inflammatory effects, antioxidant, nephroprotective, anti-apoptotic properties and so on [11-17]. Previous studies found that Cordyceps sinensis extract (CSE) had obvious protective effect on cerebral ischemic rats caused by middle cerebral artery occlusion (MCAO) [18], the specific effective parts and its treatment mechanism are unclear however.

 
2. The hypothesis

Estrogen has extensive biological effects on bone metabolism, lipid metabolism, cardiovascular system, nervous system and so on, which is an important hormone that regulates multiple tissue and functions in the body [19,20]. Classically, estrogen is considered a “reproductive” hormone because of its well-known role in feedback signaling in the hypothalamic-pituitary-ovarian axis. Many epidemiological investigation and animal experiments confirmed that estrogen can protect cerebral ischemic injury, however, the long-term use of estrogen can increase the risk of breast cancer and endometrial carcinoma [21,22]. As a result, people turned to plant ingredients and put forward the concept of phytoestrogens. Phytoestrogens is a kind of compound which can play a weak estrogen effect through a low affinity with steroid estrogen receptor(ER). Phytoestrogens is the most promising drug to prevent and treat ischemic stroke.
 

The isoflavones in CS belongs to phytoestrogens and it has estrogen-like activity [23]. What’s more, previous studies found that CSE had obvious protective effect on cerebral ischemic rats caused by MCAO [18]. Based on the theory of that estrogen can treat ischemic diseases; we conjecture that it is isoflavone in CS that plays the important role of treating focal cerebral ischemic/reperfusion injury.

We also guess that CSIF can influence the pathological pathway of NMDA (N-methyl-D-aspartate) activated by DAPK1 (death-associated protein kinase 1) and regulate the relevant factors in the NF-κB signaling pathway, both of which are underlying molecular mechanism. However, all of these are worthy to be explored deeply.

 
3. The establishment condition of the hypothesis
3.1 Cerebral ischemic injury induces the pathological pathway of DAPK1-NMDAR

​
Glutamate is the major excitatory transmitter in the mammalian central nervous system (CNS) and plays an essential role in neural development, excitatory synaptic transmission, and plasticity [24]. N-methyl-D-aspartate (NMDA) receptors constitute a major subtype of glutamate receptors and normally participate in rapid excitatory synaptic transmission. NMDA receptor toxicity is dependent on extracellular Ca2+, and reflects a large amount of Ca2+ influx directly through the receptor-gated ion channels [25,26]. Namely, it plays a key role in mediating glutamate excitotoxicity because of its high calcium permeability [27-29]. As a subtype of glutamate receptor, NMDA receptor links multiple intracellular catabolic processes responsible for irreversible neuronal death. NR2B, a kind of NMDA receptor subunit protein, is considered as the main type of functional NMDA receptor channel in CNS neurons [30].

Although over stimulation of NMDA receptors contributes to ischemic neuronal death, blocking them totally could be deleterious to animals and humans because targeting these receptors also blocks the physiological action of the receptor as well. Tu W. et al. reported that DAPK1, or death-associated protein kinase 1, acts as a specific ‘‘cell death signal’’ that couples NMDA receptor channels at extra-synaptic sites to ischemic neuronal death [31]. They also demonstrated that uncoupling of an activated DAPK1 from the NMDA receptor complex protects against brain damage in stroke without affecting the physiological actions of the NMDA receptors. Thus, targeting DAPK1-NMDA receptor interaction can be considered as a practical strategy for stroke therapy [31].

 
3.2 Neuroprotective effect of estrogen/CSIF

 Estrogen is an important hormone that regulates multiple tissues and functions in the body. Classically, estrogen is considered a “reproductive” hormone because of its well-known role in feedback signaling in the hypothalamic-pituitary-ovarian axis. However, studies on estrogen in the last decades confirmed its neuroprotective effects in the CNS [32]. In addition, it has been reported that estrogen causes protection against brain injury and neurodegenerative diseases; prescription of estrogen decreases brain injury after focal ischemia [33,34]. Many epidemiological investigations and animal experiments confirmed that estrogen can protect cerebral ischemic injury, but the long-term use of estrogen can increase the risk of breast cancer and endometrial carcinoma [35]. Thus people turned to plant ingredients, and put forward the concept of phytoestrogens, which are polyphenols similar to human estrogens found in plants or derived from plant precursors [36]. Phytoestrogens can reduce the morbidity of cardio-cerebrovascular disease. The functions of the endometrium and breast again are unaffected however. So phytoestrogens is considered as the most promising drug preventiing and treating the ischemic stroke [37]. Isoflavones, which are mostly derived from soy foods, are a major class of phytoestrogens that have attracted considerable attention as natural alternatives to hormone therapy [38]. Is flavones can bind to estrogen receptors and exert estrogen agonist or antagonist properties similar to those of SERMs (Selective Estrogen Receptor Modulators Serm) [39,40]. It have beneficial effects by lowering liver or blood triglyceride, total and LDL cholesterol levels, enhancing HDL cholesterol and the ratio of HDL/LDL[41] There are a lot of isoflavone components in Cordyceps sinensis[42-44]. Many research groups have reported that the effects of isoflavones obtained from Cordyceps sinensis and demonstrated that CSIF has significant estrogenic effects in OVX rats [23]. As a result, we predict boldly that CSIF is the key element of the treatment of brain injury because of its estrogenic activity.

 
3.3 GPR30 and estrogen

 Classically, estrogen can bind to two kinds of nuclear receptors, namely, estrogen receptor α (ERα) and estrogen receptor β (ERβ), and produce physiological and neuroprotective effects. Aside from nuclear receptors, estrogen has one membrane receptor, which can either be G-protein-coupled receptor 30 (GPR30), Gq-mER, or ER-X. The GPR-30 receptor is reported to be a novel estrogen receptor uniquely localized to the endoplasmic reticulum [45]. This receptor is widely distribute and has numerous physiologic or pathologic functions in the CNS [46,47]. NMDA exposure clearly promotes NR2B subunit phosphorylation at Ser-1303 and causes neuronal cell death. GPR30 mediates rapid non-genomic effects to protect neurous against injury by inhibiting p-DAPK1 dephosphorylation, which inhibits NR2B subunit phosphorylation at Ser-1303. In addition, NMDA exposure and global ischemia activate the autophagy pathway and induce cell death, which are markedly blocked by the NR2B antagonist Ro 25-6981[48]. As a result, the GPR30 receptor may act together with intracellular estrogen receptors to activate cell-signaling pathways to promote neuron survival after global ischemia [49,50]

Figure 1. Schematic of GPR30-mediated signaling.

4. Consequences of the hypothesis and discussion

We have studied the protective effect of CSE on middle cerebral artery block limitations caused by cerebral ischemia rats. But the effective constituent and mechanism of action are unclear.
Studies have shown that GPR30 played fast non genomic effects through combining with estrogen and mediating the second messenger, further inhibited the phosphorylation of DAPK1. Thereby the phosphorylation of NR2B subtypes Ser1303 sites was impeded, and the opening rate of NMDA receptors was decreased. As a result, estrogen has a significant effect on the occurrence and development of cerebrovascular diseases caused by excessive or sustained activation of NMDA receptor. Based on the above theoretical basis, we hypothesized that CSIF played a role for the treatment of brain injury through this mechanism. The theory is proposed for the first time and has an enormous significance to confirm.

Figure 2. The main idea of our hypotheses (Outside of the wire frame was confirmed; inside of the wire frame is to be confirmed)

This work was supported by National Natural Science Foundation of China (81373780).

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