Introduction
Fibromyalgia (FM) is characterized by generalized tenderness in ≥11 of 18 tender points (1) and chronic widespread pain that lasts >3 months (2). Mechanical hyperalgesia is a common symptom of FM (3), which is a painful syndrome of largely unknown etiology and pathology that is often accompanied by various phenomena. In addition, emerging evidence has indicated that pain amplification within the central nervous system serves an important role in the pathology of FM-associated pain (4), which is associated with numerous other symptoms (5). The symptoms of this painful syndrome include fatigue (6), anxiety (7), sleep disturbance (8), temporomandibular disease and depression (9). An animal model of FM must include widespread pain and associated symptoms of fatigue and psychological disturbance (3).
There are numerous animal models of FM pain, which are induced by either intramuscular injection of acidic saline (10), vagotomy (11), sound stress (12) or depletion of biogenic amines (13). A previous study described a novel generalized chronic pain or FM-like mouse model as part of a pharmacological study. This FM model was induced by intermittent cold stress (ICS), which is useful for inducing abnormal pain (14,15). Xu et al previously revealed that complex interactions exist between pain and depression (16). Previous studies have suggested that FM is associated with emotional disorders, including depression (17); ≥30% of patients with chronic pain have major depression, and 30% are diagnosed with panic and diffuse anxiety disorder (18).
Brain-derived neurotrophic factor (BDNF) is an upstream activator and a downstream target of cAMP response element-binding protein (CREB)-mediated signaling (19) CREB activity is tightly regulated by its phosphorylation at serine 133 (20); therefore, it would be useful to analyze BDNF and phosphorylated (p)-CREB levels following identical treatments (21). Furthermore, the BDNF/tropomyosin receptor kinase B-mediated signaling pathway within the spinal cord may be involved in the induction of neuropathic pain. In a previous study, treatment with a selective N-methyl-D-aspartate (NMDA)-2B receptor antagonist completely blocked BDNF-induced mechanical allodynia in animals (22). BDNF may play a protective role in FM, including pain modulation and mental disorders, such as depression. In addition, it has been suggested that the expression of BDNF is a downstream target of various antidepressants (23) and BDNF is an important candidate gene in antidepressant medication (24). In addition, the principal treatment for depression consists of pharmacotherapy with selective serotonin reuptake inhibitors (25), including Cymbalta (duloxetine), which is a Food and Drug Administration-approved drug for the treatment of FM (26).
Valeriana fauriei Briq. (VF) is a perennial herb found in all of North America, most parts of Europe, and Northern Asia (27). The genus Valeriana contains >250 species with many subspecies containing medicinal plants (28). VF has been used for many years in China and Korea (29), and contains 150–200 chemical constituents of biologically active components that can be divided into volatile oils, epoxy iridoid esters and alkaloids (30). These components have been reported to inhibit γ-aminobutyric acid (GABA) re-uptake (31) The genus Valeriana has been widely used in popular medicine for centuries to treat sleep disorders, anxiety and epilepsy. It can also modulate insomnia by interacting with various neurotransmitter systems (32). Previous studies have focused on the novel pharmacological effects of VF species in various human diseases. Some studies have attempted to explain the pharmacological functions of VF, particularly with regards to its neuroprotective effects in neurodegenerative diseases (33–35), and its ability to reduce pain, cyclic cramps and stress (36,37).
These previous findings have resulted in the hypothesis that VF may exert beneficial effects on FM with depression. Therefore, in the present study, the behavior of mice and the protein levels in the medial prefrontal cortex and hippocampus of a mouse model of FM were examined, in order to determine whether treatment with VF reverses any changes.
Materials and methods
Animals
Male adult C57BL/6J mice were obtained from Daehan Biolink, Co., Ltd. (Eumseong, South Korea). All groups contained between 12 and 14 mice with similar numbers of males: the 'control' (n=12 males) group; the drug non-treated model group 'FMS' (n=14 males); and the VF administration model group (n=14 males). Mice were housed in clear cages with access to free food and water, and were maintained in an environment with the temperature-controlled to 23±2°C, humidity-controlled to 50–60% and under a 12-h light-dark cycle (lights were turned on at 6:30 a.m.). All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the US National Institutes of Health (38). The present study was approved by the Animal Care and Use Committee of Soonchunhyang University (Cheonan, South Korea; SCH16-0062).
Drugs and treatments
VF extract was purchased from Yunpung (Eumsung, Chungbuk, South Korea), and the specimens were identified taxonomically by an Oriental medicine physician at the National Institute of Horticultural and Herbal Science, Rural Development Administration (Eumsung, South Korea). The voucher specimen (HPR-207) was deposited in the herbarium of the Herbal Crop Research Institute (Eumsung, South Korea). VF was dissolved in tap water to a concentration of 100 mg/kg/day. The animals were orally administered VF solution during stress for 24 days (39).
Experimental model of FM
An animal model of FM was constructed as previously described, with slight modification (40,41). For the chronic restraint stress (CRS) and intermittent cold stress (ICS) paradigm, the mice were restrained for 6 h (between 12 a.m. and 6 p.m.) daily in well-ventilated 50-ml conical tubes and were deprived of food and water. Control mice remained undisturbed in their cages. The protocol was scheduled for 21 days. For the ICS paradigm, mice were placed on a stainless steel mesh plate in a cold room at 4°C overnight (between 4:30 p.m. and 10:00 a.m.), followed by ICS with experimental temperatures alternating between 24 and 4°C every 30 min, between 10:00 a.m. and 4:30 p.m. These procedures were repeated for 2 days. On day 3, the mice were adapted to 24°C for 1 h prior to behavioral analysis. Control mice were maintained at 24°C for the 3 days (from 4:30 p.m. on day 1 to 10:00 a.m. on day 3) (15).
Behavioral analysis
Behavioral assessments were conducted 1 day after the final day of ICS (n=10 mice/group). Mice were allowed to acclimate to the testing room for ≥1 h prior to the assessments. Tests for nociception and depression were performed. A tail flick test (TFT), a plantar test (PTL), and the von Frey test paw withdrawal threshold (PWT) test were used to assess nociception. Subsequently, a tail suspension test (TST) was used to assess depression. For these thermal and mechanical tests, thresholds were determined from three repeated challenges at 15 min intervals, and the averages were used for statistical analysis.
TFT
A TFT is used as a test of pain response, which can be used to assess the antinociceptive effects of drugs by measuring the latency time from the onset of radiant heat exposure to withdrawal of the tail (42,43). The thermal pain threshold of the tail was measured using tail flick apparatus (IITC Life Science Inc., Woodland Hills, CA, USA). Each animal was gently restrained under a 50-ml conical tube with light manual pressure so as to minimize stress. Radiant heat was applied to the tail (2 cm distal part) and latency (seconds) was determined as the time taken for the tail to flick away from the radiant heat source. A cutoff time of 20 sec of radiant heat application was applied to avoid tissue damage to the tail. Each mouse was tested three times at each time-point, with an interval of 15 min between replicates. The average of three replicates was calculated to obtain tail withdrawal latency.
PTL
In the thermal paw withdrawal test, nociceptive threshold is assessed by determining the latency of paw withdrawal upon thermal stimulus (44,45). Mice were placed in individual clear plastic chambers on top of a glass sheet and were acclimated for 1 h. Radiant heat (IITC Life Science Inc.) was positioned under the glass sheet, and the focus of the projection bulb was aimed exactly at the plantar surface of the hind paw of the animal. Paw withdrawal latency (PWL), defined as the first occurrence of licking the hind paw, was scored. Each mouse was tested three times at each time-point, with an interval of 15 min between replicates. The average of three replicates was calculated to obtain PWL.
Von Frey PWT test
The pressure required to induce a flexor response was defined as the pain threshold. The Von Frey PWT test was conducted using digital von Frey apparatus (Aesthesiometer; IITC life Sciences Inc.), as previously reported (46,47). Mice were placed in a plastic chamber on a wire mesh grid floor and were allowed to acclimate for 1 h. In this experiment, the threshold of a given pressure with a rigid tip used to induce paw withdrawal behavior was evaluated. To prevent tissue damage, the interval time was set at at least 5 min for each paw.
TST
The TST is a widely used model for the assessment of depression-like behaviors in mice. In this test, mice are subjected to short-term, inescapable stress by being suspended by the tail resulting in the development of immobility. In the present study, the total duration of tail suspension-induced immobility was measured according to Steru et al (44). Mice were suspended 50 cm above the floor using adhesive tape placed ~1 cm from the tip of the tail and the total duration of immobility during a 6-min period was measured.
Measurement of corticosterone
Following completion of the behavioral analyses, the mice were placed in 50-ml conical tubes for 60 min (n=6 mice/group). The animals for analysis were sacrificed by decapitation using a guillotine using a decapicone (48). Trunk blood was immediately collected in plastic tubes and was centrifuged at 15,814 × g at room temperature; the serum was placed in a fresh tube. Serum corticosterone levels were determined by immunoassay using the Cortisol ELISA kit (cat no. 500360; Cayman Chemical, Ann Arbor, MI, USA) Assays were conducted according to the manufacturer's protocol.
Western blot analysis
mPFC and hippocampal tissues were lysed in mixture solution as radioimmunoprecipitation assay buffer [RIPA and EBA-1149 (Elpis Biotech, Inc., Daejeon, South Korea) and leupeptin and sodium fluoride (cat. nos. 103476-89-7 and 7681-49-4, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany], and were centrifuged at 18,341 × g for 10 min at 4°C (n=6–7 mice/group). Protein concentration was calculated using a standard Bradford protein assay. These samples (100 μg) were separated by 15% SDS-PAGE and were transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). After blocking with 5% skim milk for 1h at room temperature, the membranes were probed with the following antibodies overnight at 4°C: Anti-p-CREB (1:1,000; #9198), anti-CREB (1:1,000; #9197) (both from Cell Signaling Technology, Inc., Danvers, MA, USA), anti-BDNF (1:3,000; ab108319; Abcam, Cambridge, UK) and anti-β-tubulin (1:3,000; MA5-16308; Thermo Fisher Scientific, Inc., Rockford, IL, USA). Subsequently, the membrane was incubated with peroxidase-conjugated anti-mouse secondary antibody (1:10,000; A9044; Sigma-Aldrich; Merck KGaA) and goat anti-rabbit IgG-HRP antibody (1:5,000; LF-SA8002; Abfrontier, Seoul, Korea) for 1 h at room temperature. Immunoreactive bands were detected using an enhanced chemiluminescence kit (Elpis Biotech, Inc.). Semi-quantitative analysis of p-CREB, CREB, BDNF and β-tubulin protein expression was conducted using ImageJ software version 1.51k (National Institutes of Health, Bethesda, MD, USA).
Immunohistochemistry
The mice for immunohistochemical analysis had been anaesthetized with diethyl ether and were perfused through the left cardiac ventricle with 4% paraformaldehyde (n=4–5 mice/group). The fixed brains were removed, frozen and cut into 30-μm sections (n=4 mice/group). Frozen sections from the hippocampus were treated with 0.3% hydrogen peroxide for 5 min, blocked with normal horse serum (S-2000; Vector Laboratories, Inc., Burlingame, CA, USA), and were incubated with anti-p-CREB (1:800; #9198), anti-CREB (1:800; #9197) (both Cell Signaling Technology, Inc.). Subsequently, sections were incubated with Cy3-conjugated anti-rabbit (111-165-003) and anti-mouse (715-545-151) secondary antibodies (1:500; Jackson ImmunoResearch Laboratories, Inc., west Grove, PA, USA). After washing in PBS, sections were stained with DAPI to identify nuclei. Fluorescent images were captured using a confocal laser-scanning microscope (FV10-ASW; Olympus Corporation, Tokyo, Japan), and images were semi-quantified with ImageJ software version 1.51k using a protocol described previously with slight modifications (49).
Statistical analysis
Data are expressed as the mean ± standard error of the mean, and assessed using one-way analysis of variance (ANOVA) with subsequent Tukey's tests. All statistical analyses were performed using IBM SPSS Statistics 19 software (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.
Results
Tail-flick latency
Tail-flick latency was significantly different among the control, FM and VF-administered FM groups (Fig. 1). The FM group exhibited decreased tail-flick latency compared with the control group (Fig. 1). Conversely, the FM-induced decrease in tail-flick latency was attenuated following VF administration (Fig. 1).
PWL
Significant differences among the control, FM and VF-administered FM groups were detected with regards to the PWL of both paws (Fig. 2). The FM group exhibited decreased PWL compared with the control group (Fig. 2). Conversely, this FM-induced decrease in PWL in the plantar test was attenuated following VF administration (Fig. 2).
PWT
VF exerted beneficial effects on the PWT of both paws in the VF-administered FM group (Fig. 3). The FM group exhibited a decreased PWT compared with the control group (Fig. 3). Conversely, this FM-induced decrease in PWT was attenuated following VF administration (P<0.01; Fig. 3).
Tail suspension
The duration of immobility was measured in the TST, in order to evaluate stress-associated depression in mice. The duration of immobility in the FM group was significantly increased compared with the control group (P<0.05; Fig. 4). Following VF administration, the duration of immobility was significantly decreased compared with the FM group (Fig. 4), thus suggesting that VF may reverse depression in the FM group.
Corticosterone levels
Corticosterone levels were increased in the FM group (Fig. 5) compared with those in the control group. However, the corticosterone levels in the VF-administered FM group returned to the control levels (P<0.001; Fig. 5).
Western blot analysis and immunohistochemistry
In order to investigate whether the BDNF-CREB pathway, which is known to be associated with depression and pain, is involved in behavioral abnormalities in the FM group, western blotting (Figs. 6 and 7) was performed on samples from the medial prefrontal cortex and hippocampus. In addition, immunohistochemical analyses (Fig. 8) were performed on samples from the hippocampus of the control, FM and VF-administered FM groups. Western blot analysis revealed that the protein expression levels of BDNF and p-CREB in the medial prefrontal cortex and hippocampus were significantly reduced in the FM group compared with in the control group (P<0.05). However, these alterations were reversed by VF administration (P<0.05; Figs. 6 and 7). In addition, p-CREB was differentially expressed in the immunofluorescent-stained brain images of the control, FM and VF-administered FM groups (P<0.05; Fig. 8).
Discussion
In order to evaluate the extent to which treatment with VF alters behavioral activity and protein expression that may be associated with the pathophysiology of FM, the present study determined the effects of VF treatment on behavioral phenotypes using the TST, TFT, PWL and PWT. The present study demonstrated that BDNF and p-CREB expression were significantly decreased in the medial prefrontal cortex and hippocampus of mice in the FM group. The protein expression levels of BDNF and p-CREB were increased in the VF-administered FM model, as determined by western blotting and immunohistochemistry. In addition, mice in the FM model group exhibited a transient increase in serum corticosterone levels. These results provide evidence of a possible association between the BDNF-CREB pathway and FM susceptibility and pathophysiology.
Previous interest in herbal medicine has focused on the mechanisms underlying neuroendocrinological abnormalities, including the hypothalamic-pituitary-adrenal axis, cortisol production and BDNF, as well as impaired endogenous opioid function, alterations in GABAergic and/or glutamatergic transmission, cytokine or steroidal alterations, and abnormal circadian rhythm (50,51). It has been previously reported that BDNF, a key protein in the BDNF pathway, is a member of the neurotrophin family, which includes nerve growth factor, neurotrophin (NT)-3, NT-4 and BDNF (52). BDNF is involved in the development and survival of neurons and in modulating the activity of neurotransmitter systems (53), particularly serotonin and dopamine (52), which are abnormal in FM (54). Furthermore, increased BDNF-related hyperalgesia is dependent on an NMDA receptor-mediated mechanism (55). This study has reported that BDNF produces an acute, dose-dependent thermal hyperalgesic response in normal mice while antisense directed against either BDNF or its receptor, prevent inflammation-induced hyperalgesia. Furthermore, our FM model showed a thermal hyperalgesic response with decreased BDNF expression. There is growing evidence indicating that BDNF also serves a role in major depressive disorders and that antidepressant treatments increase serum BDNF levels (56,57). In animal and human studies, antidepressant treatments may increase central, as well as peripheral, BDNF levels (23,24). Liu et al reviewed studies that had been performed to identify additional modes of action of various herbal medicines on several mood disorders and antidepressants (57). The results suggested that Fuzi total alkaloid increased the phosphorylation levels of CREB and the expression of BDNF in the frontal cortex and hippocampus of an animal model. In addition, in murine models it was reported that the Fuzi total alkaloid-induced generation of antidepressant-like effects may involve the CREB-BDNF pathway (58). Furthermore, hydrophilic constituents of Morinda citiforlia are well known in folk medicine for a wide range of health purposes, including anti-inflammatory, antioxidative, detoxifying and cell-rejuvenating properties (59). In the present study, it was suggested that the BDNF-CREB pathway may be associated with FM-related pain. In addition, VF was revealed to exert anti-depressive and anti-hyperalgesic effects via the BDNF-CREB pathway.
Several studies have indicated that herbal hypnotics and sedatives, including Valeriana spp. (valerian) and Humulus lupulus (hops), usually work via modulation of adenosine receptors and via melatoninergic effects (60–62). In a previous study, valerian and its primary active component, valerenic acid, produced anxiolytic and sedative effects mainly via GABA-ergic mechanisms, similar to benzodiazepine drugs (62,63). Numerous studies have reported that the phenomenons of anxiety, psychological distress and depression are associated with the chronicity of pain (64,65), rather than tissue damage (66). The relationship between physical disease, psychiatric disorders and chronic pain is likely caused by a complex interaction. Ammer and Melnizky assessed the effects of pine oil and valerian on pain, sleep and tender point count in FM. This previous study indicated that valerian baths were associated with improved sleep, pine oil baths with increased sensitivity to pain in certain body areas, and plain water baths appeared to reduce pain intensity (67).
In the present study, the decrease in the protein expression levels of BDNF and p-CREB following ICS and CRS procedures was revealed to be reversed by treatment with VF. In addition, the results confirmed that the FM animal model was able to induce FM-like pain via abnormalities in BDNF signaling. The present study demonstrated the beneficial effects of VF on FM-associated symptoms, including depression and hyperalgesia. However, further research using cellular and animal model systems or human patients is required to fully characterize the pharmacological functions of VF on FM.