Effects of Hericium erinaceus Mycelium Extracts on the Functional Activity of Purinoceptors and Neuropathic Pain in Mice with L5 Spinal Nerve Ligation

Neuropathic pain is a serious clinical problem that is difficult to treat. Purinoceptors (P2Rs) transduce pain perception from the peripheral to the central nervous system and play an important role in the transmission of neuropathic pain signals. We previously found that the crude extracts of Hericium erinaceus mycelium (HE-CE) inhibited P2R-mediated signaling in cells and reduced heat-induced pain in mice. The present study explored the effects of HE-CE on neuropathic pain. We used adenosine triphosphate (ATP) as a P2R agonist to generate Ca2+ signaling and neuronal damage in a cell line. We also established a neuropathic mouse model of L5 spinal nerve ligation (L5-SNL) to examine neuropathic pain and neuroinflammation. Neuropathic pain was recorded using the von Frey test. Neuroinflammation was evaluated based on immunohistofluorescence observation of glial fibrillary acidic protein (GFAP) levels in astrocytes, ionized calcium-binding adaptor molecule1 (iba1) levels in microglia, and IL-6 levels in plasma. The results show that HE-CE and erinacine-S, but not erinacine-A, totally counteracted Ca2+ signaling and cytotoxic effects upon P2R stimulation by ATP in human osteosarcoma HOS cells and human neuroblastoma SH-SY5Y cells, respectively. SNL induced a decrease in the withdrawal pressure of the ipsilateral hind paw, indicating neuropathic pain. It also raised the GFAP level in astrocytes, the iba1 level in microglia, and the IL-6 level in plasma, indicating neuroinflammation. HE-CE significantly counteracted the SNL-induced decrease in withdrawal pressure, illustrating that it could relieve neuropathic pain. It also reduced SNL-induced increases in astrocyte GFAP levels, microglial iba1 levels, and plasma IL-6 levels, suggesting that HE-CE reduces neuroinflammation. Erinacine-S relieved neuropathic pain better than HE-CE. The present study demonstrated that HE inhibits P2R and, thus, that it can relieve neuropathic pain and neuroinflammation.


Introduction
Pain is a sensation triggered in the nervous system in response to the stimulation of the purinoceptor (P2R) by adenosine triphosphate (ATP). When cells are damaged or stressed, ATP is released from either the sensory neurons themselves [1] or from the adjacent peripheral tissue [2]. Extracellular ATP activates P2Rs in the nociceptive pathways, both at their peripheral and central terminals in the spinal cord [2][3][4][5][6].
ese P2Rs, which are categorized as including ionotropic P2X receptors (P2XRs) and metabotropic P2Y receptors (P2YRs), then generate and modulate various forms of pain [3,6,7]. Abundant evidence suggests that P2Rs are important in the transmission of neuropathic pain [7,8], which is the most debilitating of all clinical pain syndromes. Such pain results from nerve injury due to surgery, diabetes, cancer, or infection in the central or peripheral nervous system [9]. Neuropathic pain is generally resistant to currently available treatments and can be very difficult to alleviate, with only 40% of patients showing partial relief [9].
us, safe and effective treatments for relieving neuropathic pain are urgently needed. In this regard, P2R antagonists protect against neuropathic pain [10] and may therefore guide the search for analgesic medicine in patients with neuropathic pain.
Spinal nerve ligation (SNL) in rodent was first described in 1992 [11] and used as a neuropathic pain model [12][13][14][15]. SNL surgery induces severe mechanical allodynia, as evidenced by the decreased hind paw withdrawal threshold during von Frey hair stimulation. e surgery also leads to immediate postoperative pain and results in prolonged mechanical allodynia. As such, it may be a good model for studying neuropathic pain. Interleukin-6 (IL-6) is an inflammatory cytokine whose levels rise during nerve damage. SNL surgery induces spinal hypertrophy with increased expression of glial fibrillary acidic protein (GFAP) in astrocytes and of the ionized calcium-binding adaptor molecule 1 (iba1) in microglia [14], indicating activation of these cells. Activated astrocytes and microglia also play a role in the initiation and maintenance of neuropathic pain after SNL surgery [16,17]. In the present study, we explored mechanical allodynia, activation of spinal astrocytes and microglia, and plasma IL-6 levels following SNL surgery, seeking to better understand the progression of neuropathic pain. e use of natural compounds that antagonize nociceptive transmission by P2Rs has been proposed as a strategy for safe and effective relief of neuropathic pain [18]. In addition to neurotransmission, strong activation of P2R can cause Ca 2+ overload and consequent cell death [19,20]. Antagonists of P2R can rescue neurons from P2R-mediated neurotransmission and neurotoxicity [20]. We previously found that crude extracts of Hericium erinaceus (HE-CE) suppressed P2R-related Ca 2+ signaling and thermal pain [21]. Erinacine-A (E-A), a cyanthin diterpenoid, and erinacine-S (E-S), a sesterterpene, are two major components of HE-CE [22][23][24][25]. e present study investigated the inhibitory effects of HE-CE, E-A, and E-S on P2R signaling, as well as whether these substances can relieve SNL-induced neuropathic pain and neuroinflammation.

Preparation of HE-CE, E-A, and E-S.
HE-CE was prepared following the method described by Hu et al. [22][23][24]. In brief, 95% ethanol was added to the Hericium erinaceus mycelium powder and the preparation was ultrasonicated for 2 hours. e resulting solution was filtered and concentrated under a vacuum to obtain a brown extract, which was then partitioned through a 1 : 1 solution of water/ethyl acetate. e ethyl acetate layer was analyzed using silica gel column chromatography (70-230 mesh; 70 × 10 cm), and a 3 : 2 solution of n-hexane/ethyl acetate was used to perform gradient separation. E-S was obtained using rechromatography on a Sephadex LH-20 column and a silica column [24]. Finally, fractions collected using n-hexane/ethyl acetate (1 : 2) were sequentially eluted using methanol, 70% methanol through a Sephadex LH-20 column, and then 60% methanol using an RP-18 column to obtain E-A [25].

Cytosolic Free Ca 2+ Concentration ([Ca 2+ ] c )
Measurement. We measured [Ca 2+ ] c using fura-2 Ca 2+ fluorescent dye, following our previous methods [27]. In brief, cells were loaded with 10 μM fura-2 at 37°C for 40 minutes; they were then washed twice in loading buffer containing 150 mM NaCl, 5 mM KCl, 5 mM glucose, 1 mM MgCl 2 , 2.2 mM CaCl 2 , and 10 mM HEPES 10 (pH 7.4). Fluorescent measurements were performed using a dualexcitation fluorimeter (SPEX; CM systems) at 340 and 380 nm excitation and 505 nm emission. e[Ca 2+ ] c was calculated based on the fluorescence ratio between 340 nm and 380 nm excitation. Rmax was achieved by adding 0.01% digitonin to the cuvette at the end of experiments; excess EGTA was subsequently added to obtain Rmin. A Kd of 224 nM Ca 2+ was used for fura-2. Each data point represents the results of five individual experiments for each protocol, using batches of cells, and each experiment was carried out at least in duplicate.

MTT Assay.
We carried out the MTT assay following our previous method [27]. In brief, we cultured the cells in a 96-well plate with a density of 1 × 10 4 cells/well. After the treatments and washings, we added 0.4 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into the 96-well plate for 4 hours. Next, we added DMSO to dissolve the precipitate. e optical density of each well was read at 595 nm, with a reference at 650 nm, on an ELISA reader (Molecular Devices VersaMax, CA, USA). We used five batches of cells for each experiment, and each was carried out in triplicate. Center. e institutional and international standards were followed for the care of all animals (Principles of Laboratory Animal Care, NIH), and the protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the National Defense Medical Center, Taiwan. All studies involving animals are reported in accordance with the ARRIVE guidelines [28].

SNL Surgery.
We carried out L5-SNL surgery according to a previously described method for mice [14]. e mice were deeply anesthetized using 80% carbon dioxide (CO 2 )/ 20% oxygen (O 2 ) and had their dorsal hair shaved. An incision was then made to expose the left sixth lumbar spinal nerve by removing the L6 transverse process using a small scraper. e underlying fifth lumbar nerve root was then isolated and tightly ligated using 8-0 nylon thread, and the wound was sutured using muscle sutures (3-0 absorbable nylon) and skin sutures (3-0 nonabsorbable nylon). e surgical procedure in the sham group was identical, except that the fifth lumbar spinal nerve was not ligated or transected.

von Frey Test for Determining Mechanical Allodynia.
Pain reactions were evaluated using the von Frey tests, which were carried out according to a previously described method for mice [14] using an electronic von Frey apparatus (IITC Inc., CA, USA). Briefly, we placed the mice individually in a transparent acrylic box (9 × 9 × 15 cm) with a wire mesh bottom. We allowed them to acclimate to their environment for at least 30 minutes. A mechanical stimulus was then applied from underneath to the plantar aspect of the hind limb, with a gradual increase in pressure. e end point was removal of the paw, followed by clear flinching movements. After paw withdrawal, the intensity of the pressure was automatically recorded. Each test was repeated three times at intervals of 5 minutes to gain an average value. e area under the time-effect curve (AUC) was calculated for each animal according to the following formula [14]: ([paw withdrawal pressure at each time point] − [paw withdrawal pressure at preoperative baseline value]) × time (days).

Quantification of IL-6 Levels.
Blood samples were collected from the mice on day 13 following SNL surgery. Blood was mixed with 1.8 mg/mL EDTA and put on ice for less than 10 minutes. Plasma was collected by centrifugation at 1000x g for 10 minutes at 4°C and stored at −80°C until analysis. Cytokine levels were determined using a mouse IL-6 ELISA Kit (Abcam, ab100712).

Immunohistofluorescence of Spinal Astrocytes or
Microglia. Spinal cord slices were taken from L5 spinal segments in mice. Sample preparation and observation were carried out according to a previous report [14]. To detect spinal astrocytes or microglia, sample sections were incubated with either mouse monoclonal anti-GFAP antibody (Millipore) or rabbit anti-iba1 antibody (Wako), as appropriate, diluted in PBS. Following primary antibody incubation, an appropriate secondary antibody conjugated to a fluorophore was used (goat anti-mouse-IgG FITC [Millipore, Cat #AP124F] or goat anti-rabbit IgG Alexa Fluor 488). Images of sample sections were captured using a Leica DMI 6000B inverted microscope with a Leica DFC 420 camera and MetaMorph software (Major Instruments Co., Ltd.). Fluorescence from GFAP and iba1 immunopositive cells were shown within the superficial dorsal horn.

Statistical Analysis.
Statistical analysis was performed using one-way analysis of variance, and differences between treatments were assessed using Student's t-test. All p-values < 0.05 were regarded as statistically significant.

e Presence and Cytotoxicity of E-A and E-S in HE-CE.
We performed HPLC analysis to investigate the composition of HE-CE. Figure 1(a) shows the presence of E-A after about 7.4 minutes and that of E-S after about 15.1 minutes in HE-CE. Figure 1(b) shows the cytotoxicity of HE-CE, E-A, and E-S. HE-CE, E-A, and E-S did not affect the viability of human SH-SY5Y cells at concentrations below 10 μg/mL, while 25 μg/mL E-S decreased cell viability. 2+ ] c . ATP is a well-known P2R agonist, and 0.1 mM ATP induced a transient rise in [Ca 2+ ] c with a peak of approximately 195 ± 32 nM, in human HOS cells (Figure 2(b)). HE-CE and E-S individually inhibited this ATP-induced rise in [Ca 2+ ] c in HOS cells in a dose-dependent manner, with the half maximal inhibitory concentration (IC 50 ) values of 5 μg/mL and 1 μg/mL, respectively ( Figure 2). E-A exerted a small inhibitory effect on the ATP-induced rise in [Ca 2+ ] c . Specifically, at 50 μg/ mL, E-A showed an approximately 20% inhibitory effect. At 10 μg/mL, E-S almost completely blocked the ATP-induced rise in [Ca 2+ ] c at 5 μg/mL; E-S still exhibited potent blockage (approximately 75%). us, E-S was more effective than HE-CE, in this regard, and we propose that E-S is the active compound in HE-CE that inhibits the ATP-induced rise in [Ca 2+ ] c indicative of P2R-mediated signal transduction.

HE-CE Counteracted the Cellular Damage Induced by ATP.
At high concentrations, extracellular ATP causes neuronal damage [20]. We used human neuroblastoma SH-Evidence-Based Complementary and Alternative Medicine SY5Y cells to investigate the neuronal damage induced by ATP and the damage-preventing effects of ATP receptor (P2R) antagonists. Figure 3(a) shows that ATP decreased the viability of human neuroblastoma SH-SY5Y cells in a dosedependent manner. At 0.5 mM, ATP induced significant toxicity. Suramin, an antagonist of P2R, counteracted the toxicity induced by ATP at 0.5 and 1 mM. At 75 μM, suramin completely blocked the ATP-induced toxicity (Figure 3(b)). HE-CE also counteracted ATP-induced cytotoxicity in a dose-dependent manner (Figure 3(c)). At 50 μg/mL, HE-CE completely blocked both the ATP-induced toxicity (Figure 3) and the ATP-induced rise in [Ca 2+ ] c ( Figure 2).

Body
Weight of Mice following SNL Surgery. All mice survived until the 18th day of the present study. ere was no evidence of severe general toxicity until the end of this experiment. In general, body temperature remained unaltered and the animals' general health did not deteriorate when they were treated with SNL or fed with HE-CE or E-S.

SNL-Induced Neuropathic
Pain. SNL-surgery induced mechanical allodynia, as measured using the von-Frey test. Withdrawal pressure for the ipsilateral hind paw decreased from 7.94 ± 0.79 g (n � 15) to 2.36 ± 0.55 g (n � 15) following SNL surgery (Figure 5), while the withdrawal pressure remained at 7.90 ± 0.69 g (n � 5) and 7.61 ± 0.79 g in the control and sham groups, respectively. is significant decrease in withdrawal pressure (p < 0.001) in the SNL-surgery group reflected neuropathic pain, which lasted until day 40. e sham treatment did not change the ipsilateral hind paw  Evidence-Based Complementary and Alternative Medicine withdrawal pressure (Figure 5(a)), and the withdrawal pressure on the contralateral side in the SNL group remained similar to that of the control group until day 40 of this experiment ( Figure 5(b)). ese results indicate that L5-SNL induced the allodynia that prompted the ipsilateral hind paw withdrawal. SNL-induced mechanical allodynia served as a measure of neuropathic pain in the present study.

Effects of HE-CE and E-S on SNL-Induced Neuropathic
Pain. e effects of HE on neuropathic pain were investigated by measuring the withdrawal pressure of the hind paw in the von Frey test following HE-CE (100 mg/kg/day) and E-S (30 mg/kg/day) feeding. Mice fed with either HE-CE or E-S showed a significant smaller decrease in the withdrawal pressure of the ipsilateral hind paw induced by SNL. On day 13, a significant increase in withdrawal pressure was   . Surgery was carried out on day 1 following body weight measurements. Data are presented as mean ± standard deviation (5 mice/group).

Evidence-Based Complementary and Alternative Medicine
Taken together, these data show that both HE-CE and E-S have an antiallodynic effect and that E-S was significantly more effective than HE-CE in this regard (p < 0.05).

HE-CE Reduced the SNL-Induced Elevation in IL-6 Levels.
IL-6 is upregulated following nerve injury, and spinal-nerve injury induces rapid production and release of IL-6 [29]. Figure 6 shows that SNL surgery, but not sham treatment, raised the levels of IL-6 in plasma significantly. Both HE-CE and E-S significantly suppressed the increase in IL-6 following L5-SNL.  17-40 (d). Data are presented as mean ± standard deviation (n � 5 * 2). One-way analysis of variance was used to analyze the data. * * * p < 0.001 vs. sham group in (A); * p < 0.05, * * p < 0.01 vs. SNL +5% glucose group in (B); and * p < 0.05, * * p < 0.01 vs. sham group in (C) and (D). maintenance of neuropathic pain. We carried out immunohistofluorescence studies to investigate changes in astrocytes and microglia in the dorsal spinal cord (L5) on the ipsilateral side on day 13 after SNL surgery. Tissue sections were stained using GFAP-and iba1-specific antibodies, which were then visualized using appropriate fluorescentconjugated secondary antibodies to reveal the activation of astrocytes and microglia, respectively. Morphological changes in the astrocytes and microglia are shown in Figures 7 and 8, respectively. Figure 7 shows that GFAP staining, in dicating GFAP-positive astrocytes, is greatly increased after SNL surgery (Figure 7(b)), but not in the control group (Figure 7(a)) or after sham surgery (Figure 7(c)). Daily oral gavage using HE-CE suppressed this SNL-induced activation of astrocytes to the level of control group (Figure 7(d)). Activation of microglia was assessed by iba1 staining on the ipsilateral side of the dorsal spinal cord on day 13 (Figure 8). e immunofluorescence for iba1 was markedly increased in the ipsilateral L5 spinal cord dorsal horn following SNL surgery (Figure 8(b)), but not in the controls (Figure 8(a)). ere were some iba1positive microglia in the sham-treated group (Figure 8(c)), but markedly fewer than in the SNL group. Daily oral gavage with HE-CE suppressed the SNL-induced activation of microglia (Figure 8(d)) to the level seen in controls.

Both P2X4 and P2X7 Increased following SNL Surgery.
e expression levels of P2X4 and P2X7 in the spinal cord dorsal horn were observed using immunofluorescence measurements after SNL-surgery. Figure 9 shows that the levels of P2X4 and P2X7 were higher on the SNL-ipsilateral side than on the contralateral side on day 13.

Discussion
e present study demonstrated that HE-CE can reduce both L5-SNL-induced mechanical allodynia in mice and ATP-induced rises in [Ca 2+ ] c in a human cell line. e rise in [Ca 2+ ] c induced by ATP-an agonist of P2R-may reflect the cellular activity of P2Rs in pain transduction. SNL surgery causes neuropathic pain by inflicting neuronal damage on the dorsal root ganglion, which increases mechanical allodynia. e von Frey tests in the present study showed that HE-CE significantly suppresses SNL-induced allodynia, thus characterizing HE-CE as an analgesic. HE-CE also reduced the increase in IL-6 levels and the activation of astrocytes and microglia following SNL surgery, which illustrates that HE-CE can suppress neuroinflammation.
We previously reported that HE-CE inhibited P2Rcoupled Ca 2+ signaling and heat-induced pain [21]. e current study showed that HE-CE and E-S can completely inhibit the ATP-induced rise in [Ca 2+ ] c in HOS cells expressing the P2R subtypes P2X 1,4,5,6,7 R and P2Y 2,4,5,9,11 R. Our data, thus, suggest that both HE-CE and E-S inhibit P2Rs, which transduce pain from the peripheral to the central nervous system [7]. e levels of P2X4 and P2X7 in the spinal cord play important roles in the generation and maintenance of neuropathic pain [7,10,13,30]. Pharmacological blockade of spinal P2X4 receptors can relieve tactile allodynia [10]. Relatedly, α,β-methylene, a P2X4R agonist, rapidly initiates nociceptive behavior [5]. Blockage of P2X4R gene expression using an antisense oligonucleotide attenuated morphine-induced tolerance and hyperalgesia [31], while disruption of P2X7 purinoceptor gene expression blocked chronic inflammatory and neuropathic pain in mice [32]. ese findings highlight the roles of P2X4R of P2X7R in neuropathic pain. In the present study, and in our previous report [21], we provided data demonstrating that HE completely suppressed the activities of P2X 1,4,5,6,7 R and P2Y 2,4,5,9,11 R in a human cell line. We propose that the suppression of P2R function can block P2R pain transmission, conferring analgesic properties. e present study used L5-SNL surgery, which is a highly reproducible procedure that results in little surrounding tissue damage [15]. e neuropathic symptoms generated by the SNL model mimic those of human patients suffering from causalgia following a nerve injury [12]. A previous study reported that SNL mice retained neuropathic pain symptoms for 2 months [14,33]. e present study found that the SNL surgery in mice induced a high and stable level of von-Frey-measured pain behavior that persisted for 37 days. SNL-induced neuropathic pain is related to P2R activity [8]. In one study, the expression and production of spinal P2X7R increased following SNL surgery [34], while in another, the expression levels of P2X7R in the spinal cord segment were higher in the nerve surgery group than in the sham group [35,36]. Finally, P2X4 mRNA expression levels in the spinal horn increased following SNL surgery in mice [37]. e present study also found that the SNL surgery site expressed P2X4 and P2X7 at higher levels than the contralateral side ( Figure 9). Because P2X4 and P2X7 were upregulated after SNL surgery, causing chronic pain, blockage of P2XR by HE-CE and E-S might play a role in relieving SNL-induced neuropathic pain.
We found a massive elevation in plasma IL-6 levels following SNL surgery in mice. Other studies support this finding. Increases in IL-6 levels are coupled with nerve injury [38]. SNL surgery induced both spinal damage and a corresponding plasma IL-6 levels [14]. One study found that elevation in IL-6 levels is responsible for nerve injury-induced mechanical hypersensitivity and pain maintenance in rodents [38,39]. In the present study, both HE-CE and E-S suppressed the SNL-induced massive elevation in IL-6 levels ( Figure 6), illustrating the anti-inflammatory action of both substances. Some reports support our finding that HE has immunosuppressive characteristics. For example, HE can block elevated IL-6 induced by stress in mice [40]. It can also reduce lipopolysaccharide-induced IL-6 increases in both RAW264 macrophages [41] and an animal stroke model [42]. In this regard, Li proposed a possible link between relief from neuropathic pain and suppression of IL-6 levels [4] and that the suppressive effects of HE-CE and E-S on SNL-induced IL-6 elevation may explain their analgesic properties. P2X7 activation regulates the processing and release of cytokines [36], and increased P2X4R expression is coupled with neuronal inflammation in the ipsilateral spinal cord following nerve injury [37]. Since P2R blockage can suppress inflammation, we propose that HE suppresses the SNLinduced elevation in IL-6 levels that might occur by blocking P2R function through HE-CE.
In the present study, SNL surgery activated astrocytes and microglia at the site of surgery. HE-CE counteracted the elevated levels of GFAP in astrocytes and of iba1 in microglia following SNL surgery, illustrating that HE has neuroinflammation-suppressing properties. SNL surgery induces neuroinflammation and neuropathic pain by activating astrocytes and microglia, as supported by other studies. e activation of astrocytes and microglia is associated with the initiation and maintenance of neuropathic pain [14,17,43]. P2X4 and P2X7 were present in astrocytes and microglia in the present study; these receptors are known to transmit neuropathic pain [30,36]. In a previous study, chronic inflammation and neuropathic pain could not be induced and maintained when the P2X7 purinoceptor gene was disrupted [32]. e action of HE-CE as a P2R antagonist may mediate its anti-inflammatory and neuropathic pain reliever properties. P2R is a well-defined therapeutic target for inflammation and neuropathic pain [3]. e present study found that the SNL surgery side showed more P2X4 and P2X7, increased IL-6 levels, and activation of astrocytes and microglia, all of which play a role in SNL-induced neuropathic pain. HE-CE suppressed all these SNL-induced phenomena and relieved neuropathic pain. As such, HE-CE is potent to alleviate neuropathy.

Conclusions
Our data strongly indicate that HE has analgesic properties, because HE-CE suppressed L5-SNL-induced neuropathic pain and elevated plasma IL-6. Astrocyte and microglial activation may initiate and maintain neuropathic pain and neuroinflammation. HE-CE suppressed both IL-6 elevation and the activation of astrocytes and microglia following L5-SNL, and it blocked P2X4 and P2X7 activity in a human cell line. P2X4 and P2X7 are present in astrocytes and microglia, and they induce neuropathic pain and inflammation. We propose that HE-CE acts as an analgesic to relieve neuropathic pain through its properties as a P2R antagonist and anti-inflammatory.

Data Availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.

Conflicts of Interest
e authors declare that they have no conflicts of interests.