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. 2018 Dec 4;28(3):865–872. doi: 10.1007/s10068-018-0521-z

Protective role of Cordyceps militaris in Aβ1–42-induced Alzheimer’s disease in vivo

Mei Tong He 1, Ah Young Lee 1, Ji Hyun Kim 1, Chan Hum Park 2, Yu Su Shin 2, Eun Ju Cho 1,✉
PMCID: PMC6484065  PMID: 31093445

Abstract

According to the “amyloid cascade hypothesis”, amyloid-beta (Aβ) protein occupied one of the risk factors of Alzheimer’s disease (AD). Cordyceps militaris (CM) has been reported to exert anti-inflammatory, anti-oxidant, and neuroprotective activities; however, its activity against cognitive dysfunction has not been studied yet. In this study, the CM ethanol extract was administered with a dose of 100 or 200 mg/kg for 2 weeks, and behavioral assessments were performed for learning and memory function in Aβ1–42-induced AD mice models. Supplementation with CM extract enhanced new route consciousness and novel object recognition, and in the Morris water maze test, CM-administered groups showed less time to reach to the hidden platform compared with the control group. Moreover, the CM extract inhibited nitric oxide production and lipid peroxidation in the brain, liver, and kidney. The present study indicated that CM could have the protective role from cognitive impairment and progression of AD.

Keywords: Alzheimer’s disease, Amyloid beta, Cognition, Cordyceps militaris, Memory

Introduction

The occurrence of dysfunctions in neurodegenerative disease such as Alzheimer’s disease (AD) and Parkinson’s disease increases with age (Markesbery, 1997). AD is believed to be the most common cause of dementia presents with symptoms of memory loss and cognitive disorder. Although almost 1 million new AD cases per year are expected to occur by 2050 (Alzheimer’s Association, 2016), the cause of AD has not been clearly elucidated yet. According to the amyloid cascade hypothesis, deposition of amyloid-beta (Aβ) protein might lead to AD (Hardy and Higgins, 1992). Excessive accumulation of Aβ creates senile plaques (SP), which are found in AD patients’ brains, and are derived from the cleavage of amyloid precursor protein (Hardy and Higgins, 1992). It was believed that Aβ1–42 could be the main component of SP that results in AD (Zheng et al., 2013). Furthermore, accumulation of Aβ can also produce neurotoxic effects, leading to the appearance of oxidant stress and promoting microglial activation (Yan et al., 1996). Oxidative stress involves an imbalance between reactive oxygen species (ROS) and the defense system against ROS, which results in AD. Therefore, identification of protective agents against Aβ-induced oxidative stress has been in focus for treatment of AD patients. Previous studies have demonstrated that accumulation of Aβ in the brain of AD patients is the main reason for the disease, which suggested that the large amount of Aβ deposition leads to Aβ plaques are related to AD progression (Alzheimer’s Association, 2016). Since Aβ is closely connected with synaptotoxicity and cognitive dysfunction, Zheng et al. (2013) have reported that Aβ1–42-injected mice showed a wide range of neurodegenerative features in hippocampus and decreased both cytokines interferon-γ and nuclear factor-kappa B levels in the hippocampus, resulting in neurogenesis reduction. Likewise, excessive Aβ1–42 levels in the brain lead to oxidative stress and neurotoxicity, inflammation, and eventually cognitive disorder. The evidence has shown an association between Aβ and oxidative stress, which can induce an increase in ROS, excess lipid peroxidation, and formation of NO (Markesbery, 1997).

Cordyceps militaris (CM), a medicinal mushroom, was used as a folk tonic food in Asian countries since ancient times and is now widely used in pharmaceutical industries (Ma et al., 2015). Several studies have demonstrated that CM attenuates oxidative stress and protects against cancer progression and diabetes (Rao et al., 2010). CM is an entomogenous fungus with beneficial effects such as the hyperglycemia prevention, free radical scavenging activity, and anti-inflammatory effects (Hwang et al., 2008; Ma et al., 2015). In addition, CM exerts anti-inflammatory activity by nitric oxide (NO) inhibition in cellular system (Choi et al., 2012). The improvement effect of CM against memory deficits in rats treated with scopolamine was also demonstrated (Lee et al., 2011). On the basis of these reports, we proposed that CM might play a protective role against nervous system impairment in Aβ1–42-induced AD mice. Thus, in the present study, we investigated the protective effect of CM in Aβ1–42-induced AD mice models.

Materials and methods

Plant materials

The ethanol extract of CM was obtained from National Institute of Horticultural and Herbal Science (RDA, Jeollabuk-do, Korea) and was dissolved in water before use.

Reagents

Aβ1–42, donepezil, griess reagent, and malondialdehyde (MDA) were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). Trichloroacetic acid (TCA) and thiobarbituric acid (TBA) were acquired from Lancaster Synthesis (Ward Hill, MA, USA) and Kanto Chemical Co., Inc. (Tokyo, Japan), respectively. Phosphoric acid and butanol were obtained from Samchun Pure Chemical Co. Ltd (Seoul, Korea).

Experimental designs

Five-week-old male ICR mice were purchased from Orient Inc., (Gyeonggi-do, Korea), weighing 25–28 g and provided diet and water under a 12-h shift of light–dark cycle at temperature of 20 ± 2 °C and humidity of 50 ± 10%. Total 40 mice were randomized by weight and divided into the following groups (n = 8 per group) with a similar average weight at the initial stage: (1) normal group; (2) Aβ1–42-injected control group; (3) Aβ1–42 injection + CM (100 mg/kg/day) group; (4) Aβ1–42 injection + CM (200 mg/kg/day) group; (5) Aβ1–42 injection + donepezil (5 mg/kg/day) group. The CM extract and donepezil water solution were administered at doses of 100 or 200 mg/kg/day and 5 mg/kg/day, respectively. Mice in the normal group and Aβ1–42-injected control group were orally administered to water, while other groups were orally administered CM extract or donepezil for 14 days. All studies were implemented under the guidelines of the Pusan National University Institutional Animal Care and Use Committee (PNU-IACUC, approval number: PNU-2016-1383). The experimental timetable was designed as illustrated in Fig. 1.

Fig. 1.

Fig. 1

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Behavioral experimental schedule for mice injected with Aβ1–42

Aβ1–42-infused mouse model

Aβ1–42 protein was dissolved in 0.9% NaCl (1 μg/μL) and incubated at 37 °C for aggregation over 3-days before use (Li et al., 2010). Before intracerebroventricular (i.c.v) injection, mice were anaesthetized by a mixture of Zoletil 50® (30 mg/kg) and Rompun (10 mg/kg). According to the procedure established by Paxinos and Franklin (2004), Aβ1–42 protein (3 μL/3 min) was injected into the right lateral ventricle at stereotaxic coordinates (anteroposterior, − 0.2 mm; mediolateral, − 1.0 mm; dorsoventral, − 2.5 mm) taken from the atlas of the mouse brain (Park et al., 2012). The animals in the normal group received 0.9% NaCl injections and those in the other groups were injected with aggregated Aβ1–42 protein.

T-maze test

The T-maze test was implemented in accordance with the method established by Montgomery (1952). The T-formed equipment was made of black-painted boards. The maze was composed of a start box, and left arm and right arm (50 cm in length at the start and goal stems; 13 cm in width; 20 cm in height) with a clapboard to separate the two sides. In the training session, the left arm was blocked, mice were put in the start box, and a 10 min of the time required to explore the right arm exploration was recorded. After training, the mice were put back in the cages. Then, 24 h later, in the test session, the blocked left arm was opened to allow the mice to explore both left and right sides of the T-maze during 10 min. The space perceptive ability (%) was calculated as the ratio of the entry frequency in the left or right arms to the total number of arm entries multiplied by 100.

Novel object recognition test

The object recognition test (Bevins and Besheer, 2006) was completed in an open-field square apparatus (40 × 40 × 40 cm), which was black painted. For the training session, two identical objects (A, A′) were placed at fixed distances in the square apparatus. The number of touches of each object was counted 10 min after the mice were placed in the center of the square field. After a 24 h test session, the mice were placed back into the same apparatus and allowed to touch objects, with the shape of one of the objects different from what it was previously (A, B). Cognitive function was measured as object cognitive ability (%), describing the ratio of the number of touches with the familiar object (training section) or the novel object (test section) throughout the entire exploration period.

Morris water maze test

The Morris water maze test was performed using the method described by Morris (1984), with a slight modification. The laboratory apparatus consisted of a circular pool (80 cm in diameter; 40 cm in height), which was divided into four quadrants filled with water by using water-soluble non-toxic white color poster to make it invisible. Water temperature was maintained at 22 ± 2 °C. A small platform (8 cm in diameter) was set approximately 1 cm below the water surface in the center of one target quadrant at a fixed position during the training trial. Four different posters on the wall of the equipment provided visual cues for navigation. Three training trials per day were carried out for 4 days. At each training trial, mice were placed in the water facing the pool wall and allowed to swim for a latency time within 1 min to find the hidden platform. If a mouse was unable to reach the platform, it was guided smoothly to the platform and allowed to rest on the platform for few seconds. In addition, the latency time that each mouse spent in the target quadrant without the hidden platform was calculated. For the last test, the setup was filled with transparent pool water and the platform was put back into the target quadrant below 1 cm the water. The time for each mouse to arrive at the platform was recorded.

Measurement of lipid peroxidation

Lipid peroxidation measurement by assessment of MDA levels was conducted according to the method from Ohkawa et al. (1979). Soon after finishing the behavioral experiment, mice were dissected under Zoletil 50® and Rompun anaesthetization. Organs including the brain, liver, and kidney were removed immediately and stored with ice. The dissected organs were homogenized with 0.9% NaCl. The homogenate was mixed with thiobarbituric acid reacting substances (TBARS) solution, which contains 20% phosphoric acid, 46 mM TBA solution and 920 mM TCA solution. After boiling at 100 °C for 20 min, the mixed solution was ice-cooled, then 7 mL of butanol was added, and all the mixtures were centrifuged at 3000 rpm for 10 min. The absorbance values of the supernatant were measured at 540 nm while the lipid peroxidation level was calculated through the MDA standard curve.

NO scavenging activity

The method of NO production measurement was referred to Bryan and Grisham (2007). In this assessment, 150 μL of tissue homogenate was mixed with 130 μL of distilled water and 100 μL of the mixture was added with the same volume of Griess reagent. The absorbance value of the mixture was measured at 540 nm and the production of NO was substituted into the sodium nitrite (NaNO2) standard curve.

Statistical analysis

All the results were emphasized as mean ± standard deviation (SD). Statistical significance was determined using one-way analysis of variance (ANOVA), followed by Duncan’s multiple tests. The data from both the T-maze test and the novel object recognition test were assessed using Student’s t test. Statistical significance was considered at p < 0.05.

Results and discussion

Aβ is known as a key component of senile plaques found in brain; and deposits in the brain region where serving memory and cognition, especially in hippocampus. The i.c.v injection of Aβ induced neuronal death in hippocampus (Li et al., 2010). The cerebrospinal fluid is formed in lateral ventricles, so that when injecting Aβ i.c.v, it will irrigate through all brain parts (Simon and Iliff, 2016). Haughey et al. (2002b) also have reported that the lateral ventricle i.c.v with Aβ can induce spatial learning impairment. In addition, Park et al. (2012) showed increased level of Aβ in the hippocampus by i.c.v injection compared with the non-Aβ-treated group, indicating that i.c.v injection with Aβ could be activated in the hippocampus.

The T-maze test has been used to estimate the cognition of rodents for decades (Montgomery, 1952). In our experiment, the Aβ1–42-induced control group showed no significant differences between the old and new routes, indicating cognitive impairment by Aβ1–42 injection. In contrast, mice administered 100 or 200 mg/kg/day CM showed a higher percentage of entries in the new route than the old route, compared with the control group (Fig. 2). The data suggests that CM could improve spatial memory against Aβ1–42-induced cognitive disorder.

Fig. 2.

Fig. 2

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Effect of extracts from Cordyceps militaris (CM) on T-maze test. Normal = 0.9% NaCl injection + administration of water; Control = Aβ1–42 injection + administration of water; CM100 = Aβ1–42 injection + administration of CM (100 mg/kg/day); CM200 = Aβ1–42 injection + administration of CM (200 mg/kg/day); PO = Aβ1–42 injection + administration of donepezil (5 mg/kg/day). Values are mean ± SD (n = 8 per group). The letters (a, b) among groups represent significant differences (p < 0.05) by Duncan’s multiple range test and the asterisk (*) represent the significantly different of space perceptive abilities with old and new routes by Student’s t test (p < 0.05)

Generally, a novel object is more attractive than the familiar one in animals, especially rodents; hence the novel object recognition test is used to investigate the level of cognitive function (Bevins and Besheer, 2006). To study spatial object memory, we conducted the novel object recognition task in Aβ1–42-induced AD mice. The present experiment revealed that in the training session (A, A′), no significant differences were showed in object exploration among all the groups. However, after 24 h in the test section using two different objects (A, B), the normal group showed a higher significant recognition ability for the novel object than the familiar object, but the Aβ1–42-treated control group did not show differences in recognition ability. Meanwhile, mice administered CM at 100 or 200 mg/kg/day showed a significantly higher novel object cognitive ability (Fig. 3). This suggests that CM has a protective effect against object recognition impairment.

Fig. 3.

Fig. 3

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Effect of extracts from Cordyceps militaris (CM) on the results of the novel object recognition test. Normal = 0.9% NaCl injection + administration of water; Control = Aβ1–42 injection + administration of water; CM100 = Aβ1–42 injection + administration of CM (100 mg/kg/day); CM200 = Aβ1–42 injection + administration of CM (200 mg/kg/day); PO = Aβ1–42 injection + administration of donepezil (5 mg/kg/day). Values are mean ± SD (n = 8 per group). The letters (a-c) among groups represent significant differences (p < 0.05) by Duncan’s multiple range test and the asterisk (*) represent the significantly different of object recognition abilities with familiar and novel objects by Student’s t test (p < 0.05)

The Morris water maze test has been established as a reliable test for analyzing memory deficit and cognitive ability (Morris, 1984). As demonstrated in Fig. 4(A), all the groups showed a shorter time to reach the hidden platform during the learning section. After 4 days of training, both CM100- and CM200-administered groups reached the hidden platform in shorter time than the Aβ1–42-induced control group. In addition, in order to monitor the CM-related memory improvement that could be attributable to the visual and swimming ability, the latency in reaching to the exposed platform was also measured [Fig. 4(B, C)]. The results showed no significant differences in all the experimental groups, indicating that the effect of memory improvement on CM treated Aβ1–42-induced mice models was not involved in the swimming and visual abilities but the improvement of cognitive function.

Fig. 4.

Fig. 4

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Effect of extracts from Cordyceps militaris (CM) in the Morris water maze test.( A) Mice were allowed to look for the hidden platform that was invisible under water within 60 s. (B) The time spent in the hidden platform was calculated on the final day of the water maze test. (C) The time spent in the exposed platform in transparent water was calculated in the water maze test. Normal = 0.9% NaCl injection + administration of water; Control = Aβ1–42 injection + administration of water; CM100 = Aβ1–42 injection + administration of CM (100 mg/kg/day); CM200 = Aβ1–42 injection + administration of CM (200 mg/kg/day); PO = Aβ1–42 injection + administration of donepezil (5 mg/kg/day). Values are mean ± SD (n = 8 per group). The letters (a-c) among groups represent significant differences (p < 0.05) by Duncan’s multiple range test. NS: Non-significant

Several studies have evidenced that Aβ causes the oxidative stress in the liver, kidney, and skeletal muscle (Roher et al., 2009). Over-generation of ROS and NO is used to be a marker of oxidative stress. Excessive ROS levels in the brain imply apoptotic cell death, which leads to neurodegenerative diseases (Markesbery, 1997). The oxidative stress in lipid bilayers forms aldehydes that associate with lipid peroxidation. It has been elucidated that high levels of aldehydes are a toxic factor of lipid peroxidation (Butterfield et al., 2002). Previous studies have demonstrated that Aβ induced lipid peroxidation in organs (Butterfield et al., 2002). In addition, the NO-production level in the brain is also another target to indicate neuronal apoptosis (Combs et al., 2001). The AD-induced oxidative damage was attenuated by the use of natural materials such as green tea, Schisandra chinensis, and Eriobotrya japonica (Jeong et al., 2013; Kim et al., 2011). Similarly, our study also showed an increase of oxidative stress in the Aβ1–42-induced AD mouse brain, liver, and kidney. Firstly, the inhibitory effects of CM against lipid peroxidation induced by Aβ1–42 are illustrated in Table 1. MDA levels in the brain, liver, and kidney were significantly elevated by Aβ injection. Particularly, the MDA level in the brain in the normal group was 35.60 nmol/mg protein, but that in Aβ1–42-treated control group was 62.29 nmol/mg protein. However, oral administration of CM100 and CM200 attenuated MDA levels by 47.86 nmol/mg protein and 38.07 nmol/mg protein, respectively. Secondly, the effect of CM on NO generation in the brain, liver, and kidney in mice injected with Aβ1–42 was described in Table 2. NO production in brains in the normal group was 45.74 μmol/L/mg protein, while that in that Aβ1–42-induced control group was 53.61 μmol/L/mg protein. In addition, NO production reduced to 45.40 μmol/L/mg protein and 32.17 μmol/L/mg protein after administration of CM100 and CM200, respectively. The NO generation levels in mice livers also showed significant differences in the CM-administered groups. CM treatment might attenuate the NO production induced by Aβ1–42 in tissues. These results indicated that the increased level of MDA and NO production in Aβ1–42 mouse model led to oxidative stress associated with AD. Furthermore, the memory deficits and cognitive disorder induced by Aβ1–42 might be improved with CM treatment by attenuation of oxidative damage through lowering the amount of MDA and NO in tissues.

Table 1.

Effect of extracts from Cordyceps militaris (CM) on Aβ1–42-induced lipid peroxidation

Group MDA (nmol/mg protein)
Brain Liver Kidney
Normal 35.60 ± 14.61b 27.28 ± 5.32ab 57.05 ± 5.88ab
Control 62.29 ± 11.04a 30.85 ± 4.21a 63.71 ± 5.03a
CM100 47.86 ± 9.37ab 19.45 ± 8.57ab 60.30 ± 11.31ab
CM200 38.07 ± 10.08b 22.32 ± 9.53ab 62.29 ± 6.45ab
PO 38.19 ± 10.68b 18.29 ± 8.32b 51.05 ± 7.53b
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Normal = 0.9% NaCl injection + administration of water; Control = Aβ1–42 injection + administration of water; CM100 = Aβ1–42 injection + administration of CM (100 mg/kg/day); CM200 = Aβ1–42 injection + administration of CM (200 mg/kg/day); PO = Aβ1–42 injection + administration of donepezil (5 mg/kg/day). Values are presented as the mean ± SD (n = 8 per group). The different letters (a, b) indicate significant differences (p < 0.05) by Duncan’s multiple range test

Table 2.

Effect of extracts from Cordyceps militaris (CM) on Aβ1–42-induced nitric oxide formation

Group NaNO2 (µmol/mg protein)
Brain Liver Kidney
Normal 45.74 ± 5.90b 75.33 ± 23.92b 47.82 ± 13.08NS
Control 53.61 ± 6.60a 109.89 ± 29.27a 55.56 ± 7.93
CM100 45.40 ± 4.05b 71.91 ± 21.47b 39.63 ± 7.97
CM200 32.17 ± 3.43c 72.59 ± 18.71b 44.03 ± 15.94
PO 27.96 ± 1.67c 55.73 ± 9.62b 51.22 ± 17.65
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Normal = 0.9% NaCl injection + administration of water; Control = Aβ1–42 injection + administration of water; CM100 = Aβ1–42 injection + administration of CM (100 mg/kg/day); CM200 = Aβ1–42 injection + administration of CM (200 mg/kg/day); PO = Aβ1–42 injection + administration of donepezil (5 mg/kg/day). Values are presented as the mean ± SD (n = 8 per group). The different letters (a-c) indicate significant differences (p < 0.05) by Duncan’s multiple range test. NS: Non-significance

AD is the most common type of dementia, still remaining unclear fundamental cause. According to Markesbery (1997), a hypothesis of oxidative stress that can cause AD has been proposed; indicated that free radical-induced oxidative stress accelerate the process of neuron death, then to the onset of AD. In previous study, the anti-oxidative activity of CM was observed in vitro test. In addition, He et al. (2013) indicated that cordycepin, the major component of CM, has DPPH and hydroxyl radical scavenging property, which is similar to that activity of vitamin C. In addition, previous study showed that CM-treated groups exerted high cell survival rate in hippocampus under ischemic mouse model, indicating that CM has beneficial effect against neuronal cell apoptosis from oxidative damage (Hwang et al., 2008). Cordycepin, which has been known as an important bioactive compound of CM, suppressed the levels of pro-inflammatory cytokines in lipopolysaccharide-induced microglial cells (Kim et al., 2006). Also, cordycepin exerted the neuroprotective effect on oxidative damage in Aβ-induced hippocampal neurons (Song et al., 2018). We have observed the protective effects of CM against Aβ-induced neuroinflammation and apoptosis in neuronal cells and brain of mouse. In Aβ-treated C6 glial cells, CM inhibited ROS production and protected neuroinflammatory responces via the regulation of inducible nitric oxide synthase and cyclooxygenase-2 protein expression. Furthermore, the protein expression in mitogen-activated protein kinase signaling including phospho-p38 and phospho-JNK pathway indicated the reduced neuronal cell apoptosis level in neuronal cells and the Aβ-injected mouse brain tissue with CM treatment. Based on the evidences, we suggest that CM showed the protective effect on oxidative stress and neuroinflammation by regulation of inflammation- and apoptosis-related pathway.

It is unclear whether extract or compounds from CM can penetrate across blood brain barrier (BBB). However, they can affect the BBB permeability. The BBB is a defense gate to restrict the harmful and toxicity substances that invades into the brain parenchyma. However, when injury occurs, the BBB can be opened; thus, the harmful and toxicity substances move in, inducing inflammatory responses and neuronal damage (Saraiva et al., 2016). CM is known as an edible medicine for the diseases treatment. Ma et al. (2010) have identified kurarinone, a kind of flavonoids from CM, with antioxidant activity and anticancer effect. It can cross the BBB to influence the brain function (Hanrahan et al., 2015). In addition, cordycepin has protective effect on traumatic brain injury-induced BBB integrity, suggesting that it probably has the capacity of BBB penetration (Yuan et al., 2016).

In the neuron development, the neural stem cells presented in the subventricular zone (SVZ), are related to the neurogenesis (Zhang et al., 2007). The subgranular zone (SGZ) of the hippocampal dentate gyrus was also an important region linking to the memory formation. And a previous study showed that the activity of neural progenitor cells in the SGZ of the dentate gyrus were disrupted by Aβ deposition under Aβ-overexpressed amyloid precursor protein (APP) mice, which might cause memory impairment and an early onset of Alzheimer’s disease (Haughey et al., 2002a). Zhang et al. (2007) also showed the decreased neurogenesis in SVZ under Aβ-deposited APP transgenic mice. Therefore, overexpression of Aβ affects SVZ and SGZ region in the brain, which probably lead to memory impairment. In addition, cordycepin, a major component from CM, has reported to perform a preventive function in neuronal degeneration in hippocampal CA1 area and dentate gyrus region of oxygen–glucose deprivation-injured ischemia mouse, implicating the neuroprotective effect of cordycepin (Cheng et al., 2011). Therefore, based on the evidences, CM might also provide the neurogenesis effect both in the SVZ and the SGZ of the dentate gyrus in the Aβ-injected mice model.

In conclusion, we confirmed that administration of CM at doses of 100 and 200 mg/kg prevented Aβ-induced oxidative damage in the AD mouse model by obtaining evidence through behavioral experiments, inhibition of lipid peroxidation, and NO formation. As a result, CM could play a role in preventing memory deficits and learning disorder in AD. We expect that CM would be useful for prevention and treatment for the progression of memory loss and improve cognitive impairment.

Acknowledgements

This work was supported by a 2-Year Research Grant of Pusan National University.

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