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Analgesic effect of total flavonoids from Sanguis draxonis on spared nerve injury rat model of neuropathic pain
Phytomedicine 22 (2015) 1125–1132
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Analgesic effect of total flavonoids from Sanguis draxonis on spared nerve injury rat model of neuropathic pain Fu-Feng Chen a, Fu-Quan Huo b,1,∗, Hui Xiong a, Qing Wan a, Ya-Nan Zheng a, Wen-Jie Du a, Zhi-Nan Mei a,1,∗ a b
College of Pharmacy, South-Central University for Nationalities, Wuhan, Hubei 430074, PR China Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Xi’an Jiaotong University School of Medicine, Xi’an, Shanxi 710061, PR China
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Article history: Received 6 March 2015 Revised 5 August 2015 Accepted 18 August 2015
Keywords: Total flavonoids from Sanguis draxonis Spared nerve injury Neuropathic pain Astrocyte Pro-inflammatory cytokines
a b s t r a c t Background: Sanguis draxonis (SD) is a kind of red resin obtained from the wood of Dracaena cochinchinensis (Lour.) S. C. Chen (D. cochinchinensis). The active components of total flavonoids from SD (SDF) have analgesic effect. Aim: The aim of this study is to evaluate the analgesic effects and potential mechanism of SDF on mechanical hypersensitivity induced by spared nerve injury (SNI) model of neuropathic pain in the rat. Methods: SNI model in rats was established and then the rats were treated with SDF intragastric administration for 14 days. Paw withdrawal mechanical threshold (PMWT) in response to mechanical stimulation was measured by von Frey filaments on day 1 before operation and days 1, 3, 5, 7, 9, 11, 14 after operation, respectively. After 14 days, we measured the levels of nitric oxide (NO), nitric oxide synthase (NOS), tumor necrosis factor-α (TNF-α ), interleukin-1β (IL-1β ) and interleukin-10 (IL-10) in the spinal dorsal horn. In addition, the expression of fibroblast growth factor receptor 3 (FGFR3), phosphorylated cyclic AMP response elementbinding protein (p-CREB) and glial fibrillary acidic protein (GFAP) of the spinal dorsal horn was evaluated by western blotting and an immunofluorescence histochemical method, respectively. Results: Intragastric administration of SDF (100, 200, 400 mg/kg) alleviated significantly SNI-induced mechanical hypersensitivity, as PMWT increased in a dose-dependent manner. Moreover, SDF not only reduced the level of NO, NOS, TNF-α and IL-1β , but also upregulated the level of IL-10 in the spinal dorsal horn of SNI rats. At the same time, SDF (100, 200, 400 mg/kg) could inhibit the expression of FGFR3, GFAP and p-CREB in the spinal dorsal horn. Conclusion: SDF has potentially reduced mechanical hypersensitivity induced by SNI model of neuropathic pain which may be attributed to inhibition of astrocytic function (like release pro-inflammatory cytokines) and NO release as well as p-CREB activation in the spinal dorsal horn. © 2015 Elsevier GmbH. All rights reserved.
Introduction Neuropathic pain is a common chronic intractable pain, which is caused by peripheral or central nerve lesion or disease of the
Abbreviations: SD, Sanguis draxonis; D. cochinchinensis, Dracaena cochinchinensis (Lour.) S. C. Chen; SDF, total flavonoids from SD; SNI, spared nerve injury; PWMT, paw withdrawal mechanical threshold; NO, nitric oxide; NOS, nitric oxide synthase; TNFα , tumor necrosis factor-alpha; IL-1β , interleukin-1β ; IL-10, interleukin-10; FGFR3, fibroblast growth factor receptor 3; p-CREB, phosphorylated cyclic AMP response element-binding protein; GFAP, glial fibrillary acidic protein; CNS, central nervous system; DRG, dorsal root ganglion; CCI, chronic constriction injury; SNL, spinal nerve ligation; PSL, partial sciatic nerve ligation; ELISA, enzyme-linked immunosorbent assay. ∗ Corresponding author. Tel.: +86 27 67843713; fax: +86 27 67841196. E-mail addresses: [email protected] (H. Xiong), [email protected] (Z.-N. Mei). 1 These corresponding authors contributed equally to the work. http://dx.doi.org/10.1016/j.phymed.2015.08.011 0944-7113/© 2015 Elsevier GmbH. All rights reserved.
somatosensory system (Treede et al. 2008). It is characterized by the sensory abnormalities such as unpleasant abnormal sensation (dysesthesia or spontaneous pain), an increased response to painful stimuli (hyperalgesia), and pain induced by normally innocuous stimuli (allodynia) (Burakgazi et al. 2011). Since the mechanism of the neuropathic pain remains obscure, there is still a lack of effectively clinical treatments to prevent the development of neuropathic pain. Moreover, the current existing pharmacological treatment alternatives to alleviate the pain are often associated with poor efficacy and intolerable side effects (Attal et al. 2006; Dworkin et al. 2007; Honoré et al. 2011). Thus, it is a major clinical challenge to explore the effective and nontoxic analgesics for the management of neuropathic pain. Sanguis draxonis (SD) is a traditional Chinese herb also called Dragon’s Blood or Resina Draconis. It is a kind of red resin collected from natural exudates that appear in injured areas on the stem and
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branches of Dracaena cochinchinensis (Lour.) S. C. Chen (D. cochinchinensis). A variety of secondary metabolic products, such as flavonoids, saponins, and phenolic acids, exist abundantly in this plant material (Fan et al. 2008; González et al. 2004; Likhitwitayawuid et al. 2002; Nakashima et al. 2009). SD is a multi-component mixture with analgesic activity and has several therapeutic uses (Gupta et al. 2008; Zhong 2010). In addition, previous study has demonstrated that analgesic effect of SD may be partly explained on the basis of silencing pain signaling pathways caused by the inhibition of SD on capsaicininduced TRPV1 receptor currents in dorsal root ganglion (DRG) neurons and could be due to the synergistic effect of the three components of flavonoids (cochinchinenin A, cochinchinenin B, loureirin B) (Wei et al. 2013). It is predicted that total flavonoids from SD (SDF) may be the active ingredients of analgesic effect in SD. However, the potential analgesic effect of SDF in neuropathic pain still remains poorly understood. Many reports have indicated that glial cells dynamically regulate neuronal function under both physiological and pathological conditions (Temburni and Jacob 2001) and that astrocytic activation contributes to the development and maintenance of chronic pain induced by peripheral nerve injury (Tanga 2004). Once activated, proinflammatory cytokines (e.g. TNF-α , IL-1β ) released from astrocytes result in pathological pain is by acting on their receptors expressed on neurons in the pain-responsive regions of the spinal cord (Milligan and Watkins 2009). Thus, the functional inhibition of astrocytes in the spinal cord may play an important role in the effective therapy for neuropathic pain. Therefore, our current study was designed to investigate in detail the analgesic activities of SDF and explore its underlying mechanisms of mediating glia and inflammatory mediators in spared nerve injury (SNI) model of neuropathic pain. Materials and methods Drugs and reagents SD, the alcohol extract of the resinous wood of D. cochinchinensis (Family Liliaceae), was purchased from Xishuangbanna Resemblance Pharmaceutical Co., Ltd. for this study. SDF was prepared as previously described in our lab (Chen et al 2013). Briefly, SD was ground into powder and extracted with ethyl acetate twice, 2 h for each time. After filtration, the solution was combined and condensed to obtain a viscous extract (yield 35.5%, w/w). Then, the dried product was extracted twice with 0.34% NaOH for 1 h each time. Filtrate was collected and adjusted its pH to 2.0 by 10% HCl. The solution was precipitated for 12 h at room temperature. The precipitation was collected and washed with distilled water. To obtain the total flavonoids of SD, the samples were concentrated under reduced pressure at 40 °C using a vacuum evaporator at room temperature, (SDF, yield 15.9%, w/w). The content of flavonoids was 77.36% determined by the colorimetric method described by China Pharmacopeia. For qualitative determination of SDF, they were subjected to HPLC–ESI–MS/MS analysis. SDF sample were carried out in the negative ion mode by using a Thermo Scientific Ultimate 3000 HPLC system (Thermo Fisher Scientific, USA) equipped with a LPG-3400SP pump, a WPS-3000 automatic sampler injector, a VWD-3100 detector and an LCQ Series ion trap mass spectrometer detector with an ESI interface. The data were acquired and processed by Xcalibur software (Thermo Fisher Scientific, USA). The chromatographic separation was achieved at 35 °C on a Fortis Xi C18 HPLC column (4.6 mm × 250 mm, 5 μm particle sizes, Fortis, Britain). Mobile phases A and B were acetonitrile and water containing 0.1% formic acid, respectively. Gradient elution was as follows: 0–10 min, 75–70% B; 10–60 min, 70–50% B. The flow rate was 1.1 ml/min, the detection wavelength was 278 nm, and the sample injection volume was 7 μl. The operating conditions for ESI interface were as follows: negative ionization mode; spray
voltage, 4.5 kV; capillary temperature, 320 °C; nitrogen was used as the sheath gas (20 arb) and auxiliary gas (8.0 arb). The full scan range was set between m/z 100 and 2000 in the negative ion mode. Experimental animals Healthy male Sprague–Dawley rats (180–220 g) were purchased from the Tongji Medical College of Huazhong University of Science & Technology. Rats were raised under controlled room humidity (45– 75%) and temperature (22 ± 2 °C) with 12/12 h light/dark cycles and were given food and water ad libitum. Before the experiments, the rats were acclimatized to the laboratory conditions for 1 week. All the animal experimental procedures were performed in accordance with International Guidelines for Care and Use of Laboratory Animals and the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guideline, and approved by the Animal Ethical Committee of the South-Central University for Nationalities (Wuhan, PR China). All efforts were made to minimize the number of animals used and their suffering. Induction of SNI in rats and drug administration SNI rats were performed as described previously (Decosterd and Woolf 2000). Briefly, the rats were anesthetized with 10% chloral hydrate and disinfected left thigh with 75% alcohol under aseptic operation. Then incised (about 1 cm) the skin of the lateral left thigh, separated subcutaneous tissue and muscle blunt, exposed the sciatic nerve and its three branches: the sural, common peroneal and tibial nerves. The tibial and common peroneal nerves were tightly ligated with 5.0 silk and 2–3 mm of the nerve distal to the ligation was cut off. The sural nerve was left intact, and the wound was closed. At last, the incision was applied with a small amount of penicillin sodium powder. The rats were randomly divided into 6 groups (n = 16): normal control group, sham-operated group, SNI model group, SNI rats treated with SDF (400, 200, 100 mg/kg) groups. SDF was given by intragastric administration after surgery, once a day for 14 consecutive days, while the sham and SNI were given an equal volume of vehicle. Behavioral testing The rats were randomly divided into 4 groups (n = 8): normal control group, normal control treated with SDF (400, 200, 100 mg/kg) groups. The locomotor activity test was performed on 0, 30, 60 and 120 min after treated with SDF. The rats were placed in the center of the opened plastic box (40 cm × 40 cm × 35 cm) composed of black walls and a white, roughened floor and allowed to explore the environment for 10 min. The behavioral parameters (moving distance and moving time) were recorded automatically by AniLab software (AniLab Software & Instruments Co., Ltd.) during a 10-min observation period. Motor coordination performance was assessed by a rotarod apparatus with automatic timers and falling sensors (ZH-300/600, Anhui Zhenghua Bioinstrumentation Equipment Ltd., Anhui, China). All rats were given five consecutive training trials on the rotarod with an inter-trial interval of between 5 and 10 min. The accumulated time spent on the rod had to be at least 120 s to allow inclusion in the study. The rats were randomly divided into 4 groups (n = 8): normal control group, normal control treated with SDF (400, 200, 100 mg/kg) groups. The rats were given corresponding medicines, 30 min later tested by accelerating the rotarod speed from 4 rpm to 40 rpm for 5 min for 5 days. The rotarod apparatus was interfaced to a computer that collected the time each subject remained on the rod before falling off. The maximum time on the rod was 300 s (5 min). Nociceptive behavioral testing was performed on day 1 before and 1, 3, 5, 7, 9, 11 and 14 days after surgery by von Frey filaments (North
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Coast Medical Company, USA). The rat was placed in a transparent organic glass box which was placed on top of a metal mesh stand and allowed to acclimatize for a period of 30 min until cage exploration and major grooming activities had ceased. Then, used the scale of fibers (0.008 g, 0.02 g, 0.04 g, 0.7 g, 0.16 g, 0.4 g, 0.6 g, 1.0 g, 1.4 g, 2.0 g, 4.0 g, 6.0 g, 8.0 g, 10.0 g, 15.0 g, 26.0 g, 60.0 g, 100 g, 180 g, 300 g) to stimulate the lateral plantar surface of the left fourth and fifth toes of the hind paw in turn with sufficient force to cause slight bending (“C” shape) against the paw for approximately 5 s. If there was no withdrawal response, the next higher force fiber was delivered. The threshold was taken as the lowest force that evoked a brisk withdrawal response to repetitive stimuli. Baseline and post-nerve injury paw withdrawal mechanical threshold (PWMT) were calculated by averaging the threshold of three consecutive mechanical stimuli applied at least 5-min intervals. Biochemical analysis At the end of the experiment, the spinal cord of the rats was immediately removed under chloral hydrate deep anesthesia. The lumbar enlargement of the spinal cord was dissected on ice, and then was stored at −80 °C for further analysis. After the experiment, the dorsal horns of lumbar spinal cord tissues were homogenized in ice-cold physiological saline (0.9%) to make 10% (w/v) homogenates. After 10 min centrifugation at 4000 rpm at 4 °C, supernatant was collected for biochemical study. The lumbar spinal cord tissues levels of NO and NOS were determined by corresponding assay kits from Jiancheng Bioengineering Institute (Nanjing, Jiangsu Province, PR China). The lumbar spinal cord tissues levels of TNF-α , IL-1β and IL-10 were measured using corresponding enzyme-linked immunosobent assay (ELISA) kits bought from Dakewe Biotechnology Company (Shenzhen, PR China). To perform the western blot analysis of FGFR3, GFAP and p-CREB, we homogenized the dorsal horns of lumbar spinal cord tissues in a cold Radio Immunoprecipitation Assay lysis buffer (Beyotime Corporation, China) containing a 1% protease-inhibitor cocktail (SigmaAldrich, USA), followed by centrifuging at 14000 rpm for 10 min at 4 °C. A BCA protein assay kit (Beyotime Corporation, China) was used to detect total protein levels for each sample. Equal amounts of protein samples (30 μg) were separated on SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After that, blots were blocked with 5% milk in Tris-buffered saline (500 mM NaCl, 20 mM Tris–HCl, pH 7.5) containing 0.05% Tween-20 for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-FGFR3 (1:1000, Boster, Wuhan, China), rabbit anti-GFAP (1:1000, Beyotime, Shanghai, China) and anti-p-CREB (1:1000, CST, Danvers, USA), respectively. These blots were further incubated with horseradish peroxidase conjugated anti-rabbit IgG antibody (1:500; CST, USA), developed in ECL solution, and visualized with X-ray film exposure (Kodak, Shanghai, China). The intensity of the specific bands was captured and analyzed using AlphaEase FC software. Immunofluorescent histochemistry At the end of the experiment, animals were deeply anesthetized transcardially perfused with saline solution followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under chloral hydrate deep anesthesia. To study the immunofluorescent histochemistry of p-CREB and GFAP, the lumbar enlargement of the spinal cord was dissected out after the perfusion and fixed in the same, fresh fixative overnight at 4 °C. Coronal sections (25 μm) were cut in a cryostat and processed for immunofluorescence staining. Spinal cord sections were first blocked with 10% goat serum for 1 h at room temperature, then incubated for 24 h at 4 °C with primary antibody: rabbit anti-GFAP (1:1000, Beyotime, China) and
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rabbit Anti-p-CREB (1:800, CST, USA), followed by incubating with Cy3-conjugated goat anti-rabbit secondary antibody (1:800, Jackson ImmunoResearch, USA) for 2 h at room temperature. The stained results were examined with a Leica fluorescence microscope, and images were captured with a CCD Spot camera. After the images were captured, the optical density (OD) of GFAP or the number of p-CERB positive neurons in the same areas of the ipsilateral superficial dorsal horn (laminae I and II) was calculated and averaged across the five spinal sections for each rats. The relative value of GFAP immunodensity was expressed as percentage changes compared to the saline control. Careful focusing on all sections, only p-CERB positive neurons with obvious light emission were counted which represent the minimum number of immunopositive neurons within the sections (Huo et al. 2009; Mei et al. 2010). In the control experiments, the primary antibodies were omitted or replaced with normal guinea pig serum; no positive staining for the omitted or replaced antibodies was detected. Statistical analysis Data were presented as means ± standard deviation (SD). Differences in total observation time, as well as at each time point of each group, were statistically tested between the different groups using two-way repeated measures analysis of variance (two-way RM ANOVA) or one-way ANOVA, followed by a post-hoc multiple comparison (Bonferroni t-test) to analyze the data using SPSS software. P < 0.05 was considered to be statistically significant. Results Result of HPLC–ESI-MSn The 11 common components of flavonoids were identified from SDF by HPLC–ESI-MS/MS analysis (Fig. 1A and B). Thereinto, the contents of loureirin A, loureirin B, pterostilbene, resveratrol and 7,4ʹ-dihydroxyhomoisoflavonoid were 0.60%, 0.69%, 1.57%, 0.69% and 0.64% in SDF, respectively. At last, to evenly suspend the gel-like solid SDF in the vehicle, SDF was powdered and sieved for further experiments. Effects of SDF on PWMT in SNI rats Whether SDF could affect or not the autonomic activities and motor coordination of normal rats, we perform the locomotor activity test and the rotarod test. In the locomotor activity test, the moving distance and moving time have no difference between normal rat and normal rat treating with SDF (Fig. 2A and B). Meanwhile, in the rotarod test, there was also no difference between normal rat and normal rat treating with SDF on the time of remaining the rotarod (Fig. 2C). The effects of SDF on PWMT in SNI rats are represented in Fig. 2D. Within 14 days, there was no difference between normal rats and sham rats in PWMT. Compared to a baseline response of 60.0 ± 2.61 g measured 1 day before surgery (P < 0.01), the average PWMT of SNI group were 34.35 ± 1.31 g, 8.67 ± 1.53 g, 6.03 ± 2.71 g, 4.01 ± 2.90 g, 4.03 ± 1.83 g and 4.33 ± 1.20 g on days 3, 5, 7, 9, 11, 14 after operation (P < 0.01), respectively. Treating with SDF (100, 200, 400 mg/kg) for 14 days significantly attenuated mechanical hypersensitivity (P < 0.01) in response to von Frey filaments stimulation of the injured hind paw in a dose-dependent manner, compared to SNI group. These results suggested that SDF attenuated SNI-induced nociceptive response. Effects of SDF on FGFR3 expression of the spinal dorsal cord in SNI rats FGFR3 level was much higher in SNI group than that of sham group and normal group (P < 0.01). After treating with SDF (100, 200,
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Fig. 1. SDF was subjected to HPLC–ESI-MS/MS analysis. (A) The result of SDF for HPLC–ESI-MS/MS analysis. (B) The chemical structures of 7,4ʹ-dihydroxyflavone (1), resveratrol (2), dracorubin A (3), 7,4ʹ-dihydroxyhomoisoflavonoid (4), 2-methoxy-4,4ʹ-dihydroxydihydrochalcone (5), 2,6-dimethoxy-4,4ʹ-dihydroxydihydrochalcone (6), 7,4ʹ-dihydroxy8-methyflavane (7), 5,4ʹ-dihydroxy-7-methoxy-6-methylflavane (8), loureirin A (9), loureirin B (10), and pterostilbene(11).
Fig. 2. (A) Effects of SDF on the moving distance of the locomotor activity test in normal rats. (B) Effects of SDF on the moving time of the locomotor activity test in normal rats. (C) Effects of SDF on the time of remaining the ratarod in normal rats. (D) Effects of SDF on PWMT in SNI rats. ∗ P < 0.05, ∗∗ P < 0.01, compared to SNI rats; + P < 0.05, ++ P < 0.01, compared to sham rats. Data are presented as means ± SD.
400 mg/kg) for 14 days, FGFR3 expression was reduced in a dosedependent manner, compared with SNI model rats (Fig. 3A and B, P < 0.01). Effects of SDF on GFAP expression of the spinal dorsal cord in SNI rats SNI induced a robust astrocytic activation, indicated by GFAP upregulation in the ipsilateral spinal dorsal horn of SNI group (Fig. 4A
and B, P < 0.001). Immunohistochemistry of GFAP indicated that activated astrocytes showed hypertrophied cell bodies with thickened processes and GFAP-immunoreactive staining was enhanced. After 14 days SDF treated, SDF significantly inhibited astrocytic activationinduced by SNI especially in medium and high doses groups (Fig. 4A and B, P < 0.01). Simultaneously, in western blotting test, GFAP expression in the ipsilateral spinal dorsal cord of SNI rats was in accordance with that of immunofluorescent histochemical
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Effects of SDF on TNF-α , IL-1β and IL-10 levels of the spinal dorsal cord in SNI rats In SNI rats, we found that the levels of TNF-α and IL-1β in the spinal dorsal cord were significantly increased and the level of IL10 was dramatically decreased compared to sham group and normal group. After treating these rats with SDF at 200 and 400 mg/kg for 14 days, TNF-α levels were decreased by 16.14% (P < 0.05) and 39.28% (P < 0.01), respectively (Fig. 5A); IL-1β levels were decreased by 19.33% (P < 0.01) and 41.75%, respectively (P < 0.01) (Fig. 5B), while IL-10 levels were increased by 32.02% (P < 0.01) and 47.21% (P < 0.01), respectively (Fig. 5C). Effects of SDF on NO and NOS levels of the spinal dorsal cord in SNI rats
Fig. 3. Effects of SDF on FGFR3 expression of the spinal dorsal cord in SNI rats by western blotting. (A) The binds of FGFR3 expression in the spinal dorsal cord of all group rats. (B) Bar histograms show FGFR3 band intensity, as a ratio over FGFR3 and normalized over β -actin. ∗∗ P < 0.01, compared to SNI rats; ++ P < 0.01, compared to sham rats.
results (Fig. 4C). After treating with SDF at 200 and 400 mg/kg for 14 days, the levels of GFAP in the spinal dorsal horn were reduced dose-dependently compared to SNI rats (Fig. 4C, P < 0.01).
Fig. 6A and B represents the levels of the spinal cord NO and NOS in all groups of rats at the end of this study. In SNI rats, increased levels of NO and NOS in the spinal dorsal cord were observed. After 14-day treatment with SDF at 400 mg/kg, the NO and NOS levels were reduced dose-dependently by 21.51% (P < 0.01) and 29.84% (P < 0.01) compared to SNI group, respectively. Effects of SDF on p-CREB expression of the spinal dorsal cord in SNI rats We found that p-CREB-immunoreactive staining was significantly enhanced in the ipsilateral spinal dorsal horn of SNI group compared with the sham group and normal group (Fig. 7A and B, P < 0.001) while it was obviously reduced in a dose-dependent manner after 14 days SDF treatment (Fig. 7A and B, P < 0.01 for 100 mg/kg; P < 0.001
Fig. 4. Effects of SDF on GFAP expression of the spinal dorsal cord in SNI rats by immunofluorescent histochemisty staining and western blotting. (A) GFAP staining in the ipsilateral spinal cord in normal, sham, SNI and SNI treated with SDF (low, 100 mg/kg; medium, 200 mg/kg and 400 mg/kg) groups. The arrows show the magnified images of GFAP in rectangle box. (B) Bar histograms show the statistical analysis results of relative immunodensities of GFAP staining in laminae I and II of the ipsilateral spinal cord of the above-mentioned groups. (C) The binds of GFAP expression in the spinal dorsal cord of the above-mentioned groups. Bar histograms show GFAP band intensity, as a ratio over GFAP and normalized over GAPDH. ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to SNI rats; ++ P < 0.01, +++ P < 0.001, compared to sham rats. Scale bar, 20 μm.
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Fig. 5. Effects of SDF on TNF-α , IL-1β and IL-10 levels of the spinal dorsal cord in SNI rats by enzyme-linked immunosobent assay. (A) Effect of SDF on TNF-α level. (B) Effect of SDF on IL-1β level. (C) Effect of SDF on IL-10 level. ∗ P < 0.05, ∗∗ P < 0.01, compared to SNI rats; ++ P < 0.01, compared to sham rats.
dose-dependently the level of p-CREB in the spinal dorsal horn compared to SNI rats (Fig. 7C, P < 0.01). Discussion
Fig. 6. Effects of SDF on NO and NOS levels of the spinal dorsal cord in SNI rats. (A) Effect of SDF on NO level. (B) Effect of SDF on NOS level. ∗ P < 0.05, ∗∗ P < 0.01, compared to SNI rats; ++ P < 0.01, compared to sham rats.
for 200 and 400 mg/kg). Meanwhile, p-CREB level of western blotting in the ipsilateral spinal dorsal cord of SNI rats was in accordance with that of immunofluorescent histochemical staining results (Fig. 7C). SDF treatment at 100, 200 and 400 mg/kg could reduce
The results of the present study indicated that SNI rats showed obvious mechanical hypersensitivity in the ipsilateral affected hindpaw, PWMT significantly decreased on the second day and reached to the peak at approximately 2 weeks after the surgery. When the SNI rats treated with SDF for 14 days, mechanical hypersensitivity of SNI rats was significantly attenuated in a dose-dependent manner. Moreover, SDF had no influence on the autonomic nerve and motor nerve of normal rats by the locomotor activity test and the rotarod test, respectively. These data suggested that SDF was the main active ingredients of SD and had potential analgesic effect on neuropathic pain. Thus, we further examined the underlying analgesic mechanism of SDF on neuropathic pain. It is well known that members of FGFR family have multiple critical roles during the formation of the CNS from the stage of neural
Fig. 7. Effects of SDF on p-CREB expression of the spinal dorsal cord in SNI rats by immunofluorescent histochemisty staining and western blotting. (A) p-CREB staining in the ipsilateral spinal cord in normal, sham, SNI and SNI treated with SDF (low, 100 mg/kg; medium, 200 mg/kg and 400 mg/kg) groups. (B) Bar histograms show the statistical analysis results of p-CREB staining in laminae I and II of the ipsilateral spinal cord of the above-mentioned groups. (C) The binds of p-CREB expression in the spinal dorsal cord of the above-mentioned groups group rats. Bar histograms show p-CREB band intensity, as a ratio over p-CREB and normalized over GAPDH. ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to SNI rats; ++ P < 0.01, +++ P < 0.001, compared to sham rats. Scale bar, 20 μm.
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induction to the stage of terminal differentiation (Ford-Perriss et al. 2001). There are four FGFR genes, FGFR-1–4, and, within these, alternative splicing creates receptor isoforms with distinct specificities for particular FGFs. In normal CNS, astrocytes are active and carry out various functions. It could support, nourish neurons and regulate the external chemical environment of neurons. However, once astrocytes are activated following peripheral inflammation or nerve injury, the expression of GFAP, as astrocytic markers, was remarkably increased in the spinal cord (Gao and Ji 2010; Johansen et al. 2001; Chen et al. 2012). Pringle et al. (2003) reported that mice with a targeted deletion in the FGFR3 locus strongly up-regulate GFAP in gray matter (protoplasmic) astrocytes, implying that signaling through FGFR3 normally represses GFAP expression in vivo. Thus, to explore the possible relationship of SDF on FGFR3 and GFAP in the spinal dorsal cord of SNI rats, we performed western blotting and/or immunohistochemical staining analysis of FGFR3 and GFAP. Western blotting results showed that FGFR3 and GFAP expression was significantly increased in the spinal dorsal horn of SNI rats. Collectively, sharply increased numbers of GFAP positive astrocytes were activated in the spinal dorsal horn of SNI rats, too. After treating with SDF for 14 days, FGFR3 and GFAP expression were dose-dependently reduced in the spinal dorsal horn of SNI rats compared to vehicle treatment. These data implied a possibility that SDF contributes to FGFR3 through repressing GFAP expression to attenuate neuropathic pain. Previous many studies have provided compelling evidence that neuropathic pain pathogenesis is not only simply confined to the changes of the activity of neuronal systems, but also involves interaction among neurons, inflammatory immune and immune-like glial cells, as well as a raft of immune cell-derived inflammatory cytokines and chemokines (Austin et al. 2010). Once activated, astrocytes lead to pro-inflammatory responses with pathological effects (Watkins et al. 2003). It was well-characterized that damage to peripheral nerves, DRG and spinal cord was associated with a rapid immune response characterized by endogenous TNF-α and IL-1β , including their proteins, mRNA and receptors (Zelenka et al. 2005; Ma et al. 1998). On the contrary, IL-10 was an anti-inflammatory factor, whose action was predominantly through suppression of pro-inflammatory cytokines at both the injury site and in the spinal cord. In our study, we found that in the spinal dorsal cord of SNI rats, levels of TNF-α and IL-1β were up-regulated and that of IL-10 was decreased as compared to healthy rats. Intragastric administration of SDF for 14 days significantly reduced TNF-α and IL-1β levels and increased level of IL-10 in a dose-dependent manner in SNI rats as compared to vehicle treatment. These results indicated SDF has a strong in vivo antiinflammatory activity via up-regulating expression of IL-10 and suppressing expression of TNF-α and IL-1β to anti-neuropathic pain. On the other hand, interestingly, NO, a mediator, is released from astrocytes which are activated (Liu et al. 2000). NO is a free radical with signaling functions in the CNS and is generated endogenously from l-arginine by NOS (Garthwaite and Boulton 1995). NO participated in nociceptive signaling after inflammation, neuropathy, and trauma (Omote et al. 2001). Increased NO level and up-regulation of NOS production in the spinal cord could lead to hyperalgesia and allodynia in rats (Tao and Johns 2002). Moreover, NO could be involved in the structural plasticity of the granule cell layer in the dentate gyrus by regulating the expression of p-CREB in the hippocampus (Park et al. 2004). Thus, NO may magnify the signal of neuropathic pain by regulating the expression of p-CREB after nerve injury. Lots of evidence suggested that p-CREB plays a vital role in developing and maintaining the chronic neuropathy pain, and participates in the peripheral and central sensitization of nociceptive neurons (Miletic et al. 2002; Ma et al. 2003). In the process of neuropathic pain, pCREB can bind to specific DNA consensus sequences, including the cyclic AMP (cAMP) response element, and can regulate the expression of immediate-early genes including c-jun (Lamph et al. 1990) and c-fos (Sheng et al. 1991), and some late-effector genes (Tsukada et al.
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1987; Watson and Latchman 1995; Messersmith et al. 1998). Some studies have reported that p-CREB expression was increased in the spinal dorsal horn following multiple pain models (Hoeger-Bement and Sluka 2003; Wang et al. 2006). To explore the possible relationship of SDF on NO and p-CREB in the spinal dorsal cord of SNI rats, we performed western blot analysis and immunohistochemical analysis of p-CREB. We observed a significant elevation of NO, and NOS levels in the spinal dorsal cord of SNI rats. These abnormalities could be alleviated by SDF treatment via a dose-dependent manner. Moreover, the present results showed that p-CREB immunopositive neurons were densely distributed in the superficial dorsal horn of SNI rats. Besides, western blotting results showed that p-CREB expression was significantly increased in the spinal dorsal horn of SNI rats. After 14 days treatment, SDF could significantly reduce the numbers of pCREB positive profiles in the spinal dorsal horn compared to SNI rats. These findings supported the possibility that SDF represses the signal amplification of pro-inflammatory cytokines through inhibiting NO/p-CREB pathway in neuropathic pain. Conclusion The present study suggests that SDF has antinociceptive effect against SNI model of neuropathic pain that might partly be related to inhibit the releasing of pro-inflammatory cytokines from astrocytes by FGFR3/GFAP and NO/GFAP pathways in the spinal dorsal horn. Furthermore, it is likely that SDF could also repress the signal amplification of pro-inflammatory cytokines by inhibiting NO/p-CREB pathway in neuropathic pain. Conflict of interest The authors declare that there is no conflict of interest regarding the publication of this article. Acknowledgments This work was supported by the National Key Technology R&D Program in 12th Five Year Plan of China (No. 2012BAI27B06), Hubei Province Natural Science Foundation of China (No. 2013CFA 013) and China Postdoctoral Science Foundation (No. 2014M560760). References Attal, N., Cruccu, G., Haanpaa, M., Hansson, P., Jensen, T.S., Nurmikko, T., Sampaio, C., Sindrup, S., Wiffen, P., 2006. EFNS guidelines on pharmacological treatment of neuropathicpain. Eur. J. Neurol. 13, 1153–1169. Austin, P.J., Moalem-Taylor, G., 2010. The neuro-immune balance in neuropathic pain: involvement of inflammatory immune cells, immune-like glial cells and cytokines. J. Neuroimmunol. 229, 26–50. Burakgazi, A.Z., Messersmith, W., Vaidya, D., Hauer, P., Hoke, A., Polydefkis, M., 2011. Longitudinal assessment of oxaliplatin-induced neuropathy. Neurology 77, 980– 986. Chen, F.F., Xiong, H., Wang, J.X., Ding, X., Shu, G.W., Mei, Z.N., 2013. Antidiabetic effect of total flavonoids from Sanguis draxonis in type 2 diabetic rats. J. Ethnopharmacol. 149, 729–736. Chen, F.L., Dong, Y.L., Zhang, Z.J., Cao, D.L., Xu, J., Hui, J., Zhu, L., Gao, Y.J., 2012. Activation of astrocytes in the anterior cingulate cortex contributes to the affective component of pain in an inflammatory pain model. Brain Res. Bull. 87, 60–66. Decosterd, I., Woolf, C.J., 2000. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 87, 149–158. Dworkin, R.H., O’Connor, A.B., Backonja, M., Farrar, J.T., Finnerup, N.B., Jensen, T.S., Kalso, E.A., Loeser, J.D., Miaskowski, C., Nurmikko, T.J., Portenoy, R.K., Rice, A.S., Stacey, B.R., Treede, R.D., Turk, D.C., Wallace, M.S., Wallacem, M.S., 2007. Pharmacologic management of neuropathic pain: evidence-based recommendations. Pain 132, 237–251. Fan, L.L., Tu, P.F., He, J.X., Chen, H.B., Cai, S.Q., 2008. Microscopical study of original plant of Chinese drug “Dragon’s Blood” Dracaena cochinchinensis and distribution and constituents detection of its resin. China J. Chin. Mater. Med. 10, 003. Ford-Perriss, M., Abud, H., Murphy, M., 2001. Fibroblast growth factors in the developing central nervous system. Clin. Exp. Pharmacol. Physiol. 28, 493–503. Gao, Y.J., Ji, R.R., 2010. Targeting astrocyte signaling for chronic pain. Neurotherapeutics 7, 482–493. Garthwaite, J., Boulton, C.L., 1995. Nitric oxide signaling in the central nervous system. Annu. Rev. Pathol. 57, 683–706.
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Analgesic effect of total flavonoids from Sanguis draxonis on spared nerve injury rat model of neuropathic pain Fu-Feng Chen a, Fu-Quan Huo b,1,∗, Hui Xiong a, Qing Wan a, Ya-Nan Zheng a, Wen-Jie Du a, Zhi-Nan Mei a,1,∗ a b
College of Pharmacy, South-Central University for Nationalities, Wuhan, Hubei 430074, PR China Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Xi’an Jiaotong University School of Medicine, Xi’an, Shanxi 710061, PR China
a r t i c l e
i n f o
Article history: Received 6 March 2015 Revised 5 August 2015 Accepted 18 August 2015
Keywords: Total flavonoids from Sanguis draxonis Spared nerve injury Neuropathic pain Astrocyte Pro-inflammatory cytokines
a b s t r a c t Background: Sanguis draxonis (SD) is a kind of red resin obtained from the wood of Dracaena cochinchinensis (Lour.) S. C. Chen (D. cochinchinensis). The active components of total flavonoids from SD (SDF) have analgesic effect. Aim: The aim of this study is to evaluate the analgesic effects and potential mechanism of SDF on mechanical hypersensitivity induced by spared nerve injury (SNI) model of neuropathic pain in the rat. Methods: SNI model in rats was established and then the rats were treated with SDF intragastric administration for 14 days. Paw withdrawal mechanical threshold (PMWT) in response to mechanical stimulation was measured by von Frey filaments on day 1 before operation and days 1, 3, 5, 7, 9, 11, 14 after operation, respectively. After 14 days, we measured the levels of nitric oxide (NO), nitric oxide synthase (NOS), tumor necrosis factor-α (TNF-α ), interleukin-1β (IL-1β ) and interleukin-10 (IL-10) in the spinal dorsal horn. In addition, the expression of fibroblast growth factor receptor 3 (FGFR3), phosphorylated cyclic AMP response elementbinding protein (p-CREB) and glial fibrillary acidic protein (GFAP) of the spinal dorsal horn was evaluated by western blotting and an immunofluorescence histochemical method, respectively. Results: Intragastric administration of SDF (100, 200, 400 mg/kg) alleviated significantly SNI-induced mechanical hypersensitivity, as PMWT increased in a dose-dependent manner. Moreover, SDF not only reduced the level of NO, NOS, TNF-α and IL-1β , but also upregulated the level of IL-10 in the spinal dorsal horn of SNI rats. At the same time, SDF (100, 200, 400 mg/kg) could inhibit the expression of FGFR3, GFAP and p-CREB in the spinal dorsal horn. Conclusion: SDF has potentially reduced mechanical hypersensitivity induced by SNI model of neuropathic pain which may be attributed to inhibition of astrocytic function (like release pro-inflammatory cytokines) and NO release as well as p-CREB activation in the spinal dorsal horn. © 2015 Elsevier GmbH. All rights reserved.
Introduction Neuropathic pain is a common chronic intractable pain, which is caused by peripheral or central nerve lesion or disease of the
Abbreviations: SD, Sanguis draxonis; D. cochinchinensis, Dracaena cochinchinensis (Lour.) S. C. Chen; SDF, total flavonoids from SD; SNI, spared nerve injury; PWMT, paw withdrawal mechanical threshold; NO, nitric oxide; NOS, nitric oxide synthase; TNFα , tumor necrosis factor-alpha; IL-1β , interleukin-1β ; IL-10, interleukin-10; FGFR3, fibroblast growth factor receptor 3; p-CREB, phosphorylated cyclic AMP response element-binding protein; GFAP, glial fibrillary acidic protein; CNS, central nervous system; DRG, dorsal root ganglion; CCI, chronic constriction injury; SNL, spinal nerve ligation; PSL, partial sciatic nerve ligation; ELISA, enzyme-linked immunosorbent assay. ∗ Corresponding author. Tel.: +86 27 67843713; fax: +86 27 67841196. E-mail addresses: [email protected] (H. Xiong), [email protected] (Z.-N. Mei). 1 These corresponding authors contributed equally to the work. http://dx.doi.org/10.1016/j.phymed.2015.08.011 0944-7113/© 2015 Elsevier GmbH. All rights reserved.
somatosensory system (Treede et al. 2008). It is characterized by the sensory abnormalities such as unpleasant abnormal sensation (dysesthesia or spontaneous pain), an increased response to painful stimuli (hyperalgesia), and pain induced by normally innocuous stimuli (allodynia) (Burakgazi et al. 2011). Since the mechanism of the neuropathic pain remains obscure, there is still a lack of effectively clinical treatments to prevent the development of neuropathic pain. Moreover, the current existing pharmacological treatment alternatives to alleviate the pain are often associated with poor efficacy and intolerable side effects (Attal et al. 2006; Dworkin et al. 2007; Honoré et al. 2011). Thus, it is a major clinical challenge to explore the effective and nontoxic analgesics for the management of neuropathic pain. Sanguis draxonis (SD) is a traditional Chinese herb also called Dragon’s Blood or Resina Draconis. It is a kind of red resin collected from natural exudates that appear in injured areas on the stem and
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branches of Dracaena cochinchinensis (Lour.) S. C. Chen (D. cochinchinensis). A variety of secondary metabolic products, such as flavonoids, saponins, and phenolic acids, exist abundantly in this plant material (Fan et al. 2008; González et al. 2004; Likhitwitayawuid et al. 2002; Nakashima et al. 2009). SD is a multi-component mixture with analgesic activity and has several therapeutic uses (Gupta et al. 2008; Zhong 2010). In addition, previous study has demonstrated that analgesic effect of SD may be partly explained on the basis of silencing pain signaling pathways caused by the inhibition of SD on capsaicininduced TRPV1 receptor currents in dorsal root ganglion (DRG) neurons and could be due to the synergistic effect of the three components of flavonoids (cochinchinenin A, cochinchinenin B, loureirin B) (Wei et al. 2013). It is predicted that total flavonoids from SD (SDF) may be the active ingredients of analgesic effect in SD. However, the potential analgesic effect of SDF in neuropathic pain still remains poorly understood. Many reports have indicated that glial cells dynamically regulate neuronal function under both physiological and pathological conditions (Temburni and Jacob 2001) and that astrocytic activation contributes to the development and maintenance of chronic pain induced by peripheral nerve injury (Tanga 2004). Once activated, proinflammatory cytokines (e.g. TNF-α , IL-1β ) released from astrocytes result in pathological pain is by acting on their receptors expressed on neurons in the pain-responsive regions of the spinal cord (Milligan and Watkins 2009). Thus, the functional inhibition of astrocytes in the spinal cord may play an important role in the effective therapy for neuropathic pain. Therefore, our current study was designed to investigate in detail the analgesic activities of SDF and explore its underlying mechanisms of mediating glia and inflammatory mediators in spared nerve injury (SNI) model of neuropathic pain. Materials and methods Drugs and reagents SD, the alcohol extract of the resinous wood of D. cochinchinensis (Family Liliaceae), was purchased from Xishuangbanna Resemblance Pharmaceutical Co., Ltd. for this study. SDF was prepared as previously described in our lab (Chen et al 2013). Briefly, SD was ground into powder and extracted with ethyl acetate twice, 2 h for each time. After filtration, the solution was combined and condensed to obtain a viscous extract (yield 35.5%, w/w). Then, the dried product was extracted twice with 0.34% NaOH for 1 h each time. Filtrate was collected and adjusted its pH to 2.0 by 10% HCl. The solution was precipitated for 12 h at room temperature. The precipitation was collected and washed with distilled water. To obtain the total flavonoids of SD, the samples were concentrated under reduced pressure at 40 °C using a vacuum evaporator at room temperature, (SDF, yield 15.9%, w/w). The content of flavonoids was 77.36% determined by the colorimetric method described by China Pharmacopeia. For qualitative determination of SDF, they were subjected to HPLC–ESI–MS/MS analysis. SDF sample were carried out in the negative ion mode by using a Thermo Scientific Ultimate 3000 HPLC system (Thermo Fisher Scientific, USA) equipped with a LPG-3400SP pump, a WPS-3000 automatic sampler injector, a VWD-3100 detector and an LCQ Series ion trap mass spectrometer detector with an ESI interface. The data were acquired and processed by Xcalibur software (Thermo Fisher Scientific, USA). The chromatographic separation was achieved at 35 °C on a Fortis Xi C18 HPLC column (4.6 mm × 250 mm, 5 μm particle sizes, Fortis, Britain). Mobile phases A and B were acetonitrile and water containing 0.1% formic acid, respectively. Gradient elution was as follows: 0–10 min, 75–70% B; 10–60 min, 70–50% B. The flow rate was 1.1 ml/min, the detection wavelength was 278 nm, and the sample injection volume was 7 μl. The operating conditions for ESI interface were as follows: negative ionization mode; spray
voltage, 4.5 kV; capillary temperature, 320 °C; nitrogen was used as the sheath gas (20 arb) and auxiliary gas (8.0 arb). The full scan range was set between m/z 100 and 2000 in the negative ion mode. Experimental animals Healthy male Sprague–Dawley rats (180–220 g) were purchased from the Tongji Medical College of Huazhong University of Science & Technology. Rats were raised under controlled room humidity (45– 75%) and temperature (22 ± 2 °C) with 12/12 h light/dark cycles and were given food and water ad libitum. Before the experiments, the rats were acclimatized to the laboratory conditions for 1 week. All the animal experimental procedures were performed in accordance with International Guidelines for Care and Use of Laboratory Animals and the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guideline, and approved by the Animal Ethical Committee of the South-Central University for Nationalities (Wuhan, PR China). All efforts were made to minimize the number of animals used and their suffering. Induction of SNI in rats and drug administration SNI rats were performed as described previously (Decosterd and Woolf 2000). Briefly, the rats were anesthetized with 10% chloral hydrate and disinfected left thigh with 75% alcohol under aseptic operation. Then incised (about 1 cm) the skin of the lateral left thigh, separated subcutaneous tissue and muscle blunt, exposed the sciatic nerve and its three branches: the sural, common peroneal and tibial nerves. The tibial and common peroneal nerves were tightly ligated with 5.0 silk and 2–3 mm of the nerve distal to the ligation was cut off. The sural nerve was left intact, and the wound was closed. At last, the incision was applied with a small amount of penicillin sodium powder. The rats were randomly divided into 6 groups (n = 16): normal control group, sham-operated group, SNI model group, SNI rats treated with SDF (400, 200, 100 mg/kg) groups. SDF was given by intragastric administration after surgery, once a day for 14 consecutive days, while the sham and SNI were given an equal volume of vehicle. Behavioral testing The rats were randomly divided into 4 groups (n = 8): normal control group, normal control treated with SDF (400, 200, 100 mg/kg) groups. The locomotor activity test was performed on 0, 30, 60 and 120 min after treated with SDF. The rats were placed in the center of the opened plastic box (40 cm × 40 cm × 35 cm) composed of black walls and a white, roughened floor and allowed to explore the environment for 10 min. The behavioral parameters (moving distance and moving time) were recorded automatically by AniLab software (AniLab Software & Instruments Co., Ltd.) during a 10-min observation period. Motor coordination performance was assessed by a rotarod apparatus with automatic timers and falling sensors (ZH-300/600, Anhui Zhenghua Bioinstrumentation Equipment Ltd., Anhui, China). All rats were given five consecutive training trials on the rotarod with an inter-trial interval of between 5 and 10 min. The accumulated time spent on the rod had to be at least 120 s to allow inclusion in the study. The rats were randomly divided into 4 groups (n = 8): normal control group, normal control treated with SDF (400, 200, 100 mg/kg) groups. The rats were given corresponding medicines, 30 min later tested by accelerating the rotarod speed from 4 rpm to 40 rpm for 5 min for 5 days. The rotarod apparatus was interfaced to a computer that collected the time each subject remained on the rod before falling off. The maximum time on the rod was 300 s (5 min). Nociceptive behavioral testing was performed on day 1 before and 1, 3, 5, 7, 9, 11 and 14 days after surgery by von Frey filaments (North
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Coast Medical Company, USA). The rat was placed in a transparent organic glass box which was placed on top of a metal mesh stand and allowed to acclimatize for a period of 30 min until cage exploration and major grooming activities had ceased. Then, used the scale of fibers (0.008 g, 0.02 g, 0.04 g, 0.7 g, 0.16 g, 0.4 g, 0.6 g, 1.0 g, 1.4 g, 2.0 g, 4.0 g, 6.0 g, 8.0 g, 10.0 g, 15.0 g, 26.0 g, 60.0 g, 100 g, 180 g, 300 g) to stimulate the lateral plantar surface of the left fourth and fifth toes of the hind paw in turn with sufficient force to cause slight bending (“C” shape) against the paw for approximately 5 s. If there was no withdrawal response, the next higher force fiber was delivered. The threshold was taken as the lowest force that evoked a brisk withdrawal response to repetitive stimuli. Baseline and post-nerve injury paw withdrawal mechanical threshold (PWMT) were calculated by averaging the threshold of three consecutive mechanical stimuli applied at least 5-min intervals. Biochemical analysis At the end of the experiment, the spinal cord of the rats was immediately removed under chloral hydrate deep anesthesia. The lumbar enlargement of the spinal cord was dissected on ice, and then was stored at −80 °C for further analysis. After the experiment, the dorsal horns of lumbar spinal cord tissues were homogenized in ice-cold physiological saline (0.9%) to make 10% (w/v) homogenates. After 10 min centrifugation at 4000 rpm at 4 °C, supernatant was collected for biochemical study. The lumbar spinal cord tissues levels of NO and NOS were determined by corresponding assay kits from Jiancheng Bioengineering Institute (Nanjing, Jiangsu Province, PR China). The lumbar spinal cord tissues levels of TNF-α , IL-1β and IL-10 were measured using corresponding enzyme-linked immunosobent assay (ELISA) kits bought from Dakewe Biotechnology Company (Shenzhen, PR China). To perform the western blot analysis of FGFR3, GFAP and p-CREB, we homogenized the dorsal horns of lumbar spinal cord tissues in a cold Radio Immunoprecipitation Assay lysis buffer (Beyotime Corporation, China) containing a 1% protease-inhibitor cocktail (SigmaAldrich, USA), followed by centrifuging at 14000 rpm for 10 min at 4 °C. A BCA protein assay kit (Beyotime Corporation, China) was used to detect total protein levels for each sample. Equal amounts of protein samples (30 μg) were separated on SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After that, blots were blocked with 5% milk in Tris-buffered saline (500 mM NaCl, 20 mM Tris–HCl, pH 7.5) containing 0.05% Tween-20 for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-FGFR3 (1:1000, Boster, Wuhan, China), rabbit anti-GFAP (1:1000, Beyotime, Shanghai, China) and anti-p-CREB (1:1000, CST, Danvers, USA), respectively. These blots were further incubated with horseradish peroxidase conjugated anti-rabbit IgG antibody (1:500; CST, USA), developed in ECL solution, and visualized with X-ray film exposure (Kodak, Shanghai, China). The intensity of the specific bands was captured and analyzed using AlphaEase FC software. Immunofluorescent histochemistry At the end of the experiment, animals were deeply anesthetized transcardially perfused with saline solution followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under chloral hydrate deep anesthesia. To study the immunofluorescent histochemistry of p-CREB and GFAP, the lumbar enlargement of the spinal cord was dissected out after the perfusion and fixed in the same, fresh fixative overnight at 4 °C. Coronal sections (25 μm) were cut in a cryostat and processed for immunofluorescence staining. Spinal cord sections were first blocked with 10% goat serum for 1 h at room temperature, then incubated for 24 h at 4 °C with primary antibody: rabbit anti-GFAP (1:1000, Beyotime, China) and
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rabbit Anti-p-CREB (1:800, CST, USA), followed by incubating with Cy3-conjugated goat anti-rabbit secondary antibody (1:800, Jackson ImmunoResearch, USA) for 2 h at room temperature. The stained results were examined with a Leica fluorescence microscope, and images were captured with a CCD Spot camera. After the images were captured, the optical density (OD) of GFAP or the number of p-CERB positive neurons in the same areas of the ipsilateral superficial dorsal horn (laminae I and II) was calculated and averaged across the five spinal sections for each rats. The relative value of GFAP immunodensity was expressed as percentage changes compared to the saline control. Careful focusing on all sections, only p-CERB positive neurons with obvious light emission were counted which represent the minimum number of immunopositive neurons within the sections (Huo et al. 2009; Mei et al. 2010). In the control experiments, the primary antibodies were omitted or replaced with normal guinea pig serum; no positive staining for the omitted or replaced antibodies was detected. Statistical analysis Data were presented as means ± standard deviation (SD). Differences in total observation time, as well as at each time point of each group, were statistically tested between the different groups using two-way repeated measures analysis of variance (two-way RM ANOVA) or one-way ANOVA, followed by a post-hoc multiple comparison (Bonferroni t-test) to analyze the data using SPSS software. P < 0.05 was considered to be statistically significant. Results Result of HPLC–ESI-MSn The 11 common components of flavonoids were identified from SDF by HPLC–ESI-MS/MS analysis (Fig. 1A and B). Thereinto, the contents of loureirin A, loureirin B, pterostilbene, resveratrol and 7,4ʹ-dihydroxyhomoisoflavonoid were 0.60%, 0.69%, 1.57%, 0.69% and 0.64% in SDF, respectively. At last, to evenly suspend the gel-like solid SDF in the vehicle, SDF was powdered and sieved for further experiments. Effects of SDF on PWMT in SNI rats Whether SDF could affect or not the autonomic activities and motor coordination of normal rats, we perform the locomotor activity test and the rotarod test. In the locomotor activity test, the moving distance and moving time have no difference between normal rat and normal rat treating with SDF (Fig. 2A and B). Meanwhile, in the rotarod test, there was also no difference between normal rat and normal rat treating with SDF on the time of remaining the rotarod (Fig. 2C). The effects of SDF on PWMT in SNI rats are represented in Fig. 2D. Within 14 days, there was no difference between normal rats and sham rats in PWMT. Compared to a baseline response of 60.0 ± 2.61 g measured 1 day before surgery (P < 0.01), the average PWMT of SNI group were 34.35 ± 1.31 g, 8.67 ± 1.53 g, 6.03 ± 2.71 g, 4.01 ± 2.90 g, 4.03 ± 1.83 g and 4.33 ± 1.20 g on days 3, 5, 7, 9, 11, 14 after operation (P < 0.01), respectively. Treating with SDF (100, 200, 400 mg/kg) for 14 days significantly attenuated mechanical hypersensitivity (P < 0.01) in response to von Frey filaments stimulation of the injured hind paw in a dose-dependent manner, compared to SNI group. These results suggested that SDF attenuated SNI-induced nociceptive response. Effects of SDF on FGFR3 expression of the spinal dorsal cord in SNI rats FGFR3 level was much higher in SNI group than that of sham group and normal group (P < 0.01). After treating with SDF (100, 200,
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Fig. 1. SDF was subjected to HPLC–ESI-MS/MS analysis. (A) The result of SDF for HPLC–ESI-MS/MS analysis. (B) The chemical structures of 7,4ʹ-dihydroxyflavone (1), resveratrol (2), dracorubin A (3), 7,4ʹ-dihydroxyhomoisoflavonoid (4), 2-methoxy-4,4ʹ-dihydroxydihydrochalcone (5), 2,6-dimethoxy-4,4ʹ-dihydroxydihydrochalcone (6), 7,4ʹ-dihydroxy8-methyflavane (7), 5,4ʹ-dihydroxy-7-methoxy-6-methylflavane (8), loureirin A (9), loureirin B (10), and pterostilbene(11).
Fig. 2. (A) Effects of SDF on the moving distance of the locomotor activity test in normal rats. (B) Effects of SDF on the moving time of the locomotor activity test in normal rats. (C) Effects of SDF on the time of remaining the ratarod in normal rats. (D) Effects of SDF on PWMT in SNI rats. ∗ P < 0.05, ∗∗ P < 0.01, compared to SNI rats; + P < 0.05, ++ P < 0.01, compared to sham rats. Data are presented as means ± SD.
400 mg/kg) for 14 days, FGFR3 expression was reduced in a dosedependent manner, compared with SNI model rats (Fig. 3A and B, P < 0.01). Effects of SDF on GFAP expression of the spinal dorsal cord in SNI rats SNI induced a robust astrocytic activation, indicated by GFAP upregulation in the ipsilateral spinal dorsal horn of SNI group (Fig. 4A
and B, P < 0.001). Immunohistochemistry of GFAP indicated that activated astrocytes showed hypertrophied cell bodies with thickened processes and GFAP-immunoreactive staining was enhanced. After 14 days SDF treated, SDF significantly inhibited astrocytic activationinduced by SNI especially in medium and high doses groups (Fig. 4A and B, P < 0.01). Simultaneously, in western blotting test, GFAP expression in the ipsilateral spinal dorsal cord of SNI rats was in accordance with that of immunofluorescent histochemical
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Effects of SDF on TNF-α , IL-1β and IL-10 levels of the spinal dorsal cord in SNI rats In SNI rats, we found that the levels of TNF-α and IL-1β in the spinal dorsal cord were significantly increased and the level of IL10 was dramatically decreased compared to sham group and normal group. After treating these rats with SDF at 200 and 400 mg/kg for 14 days, TNF-α levels were decreased by 16.14% (P < 0.05) and 39.28% (P < 0.01), respectively (Fig. 5A); IL-1β levels were decreased by 19.33% (P < 0.01) and 41.75%, respectively (P < 0.01) (Fig. 5B), while IL-10 levels were increased by 32.02% (P < 0.01) and 47.21% (P < 0.01), respectively (Fig. 5C). Effects of SDF on NO and NOS levels of the spinal dorsal cord in SNI rats
Fig. 3. Effects of SDF on FGFR3 expression of the spinal dorsal cord in SNI rats by western blotting. (A) The binds of FGFR3 expression in the spinal dorsal cord of all group rats. (B) Bar histograms show FGFR3 band intensity, as a ratio over FGFR3 and normalized over β -actin. ∗∗ P < 0.01, compared to SNI rats; ++ P < 0.01, compared to sham rats.
results (Fig. 4C). After treating with SDF at 200 and 400 mg/kg for 14 days, the levels of GFAP in the spinal dorsal horn were reduced dose-dependently compared to SNI rats (Fig. 4C, P < 0.01).
Fig. 6A and B represents the levels of the spinal cord NO and NOS in all groups of rats at the end of this study. In SNI rats, increased levels of NO and NOS in the spinal dorsal cord were observed. After 14-day treatment with SDF at 400 mg/kg, the NO and NOS levels were reduced dose-dependently by 21.51% (P < 0.01) and 29.84% (P < 0.01) compared to SNI group, respectively. Effects of SDF on p-CREB expression of the spinal dorsal cord in SNI rats We found that p-CREB-immunoreactive staining was significantly enhanced in the ipsilateral spinal dorsal horn of SNI group compared with the sham group and normal group (Fig. 7A and B, P < 0.001) while it was obviously reduced in a dose-dependent manner after 14 days SDF treatment (Fig. 7A and B, P < 0.01 for 100 mg/kg; P < 0.001
Fig. 4. Effects of SDF on GFAP expression of the spinal dorsal cord in SNI rats by immunofluorescent histochemisty staining and western blotting. (A) GFAP staining in the ipsilateral spinal cord in normal, sham, SNI and SNI treated with SDF (low, 100 mg/kg; medium, 200 mg/kg and 400 mg/kg) groups. The arrows show the magnified images of GFAP in rectangle box. (B) Bar histograms show the statistical analysis results of relative immunodensities of GFAP staining in laminae I and II of the ipsilateral spinal cord of the above-mentioned groups. (C) The binds of GFAP expression in the spinal dorsal cord of the above-mentioned groups. Bar histograms show GFAP band intensity, as a ratio over GFAP and normalized over GAPDH. ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to SNI rats; ++ P < 0.01, +++ P < 0.001, compared to sham rats. Scale bar, 20 μm.
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Fig. 5. Effects of SDF on TNF-α , IL-1β and IL-10 levels of the spinal dorsal cord in SNI rats by enzyme-linked immunosobent assay. (A) Effect of SDF on TNF-α level. (B) Effect of SDF on IL-1β level. (C) Effect of SDF on IL-10 level. ∗ P < 0.05, ∗∗ P < 0.01, compared to SNI rats; ++ P < 0.01, compared to sham rats.
dose-dependently the level of p-CREB in the spinal dorsal horn compared to SNI rats (Fig. 7C, P < 0.01). Discussion
Fig. 6. Effects of SDF on NO and NOS levels of the spinal dorsal cord in SNI rats. (A) Effect of SDF on NO level. (B) Effect of SDF on NOS level. ∗ P < 0.05, ∗∗ P < 0.01, compared to SNI rats; ++ P < 0.01, compared to sham rats.
for 200 and 400 mg/kg). Meanwhile, p-CREB level of western blotting in the ipsilateral spinal dorsal cord of SNI rats was in accordance with that of immunofluorescent histochemical staining results (Fig. 7C). SDF treatment at 100, 200 and 400 mg/kg could reduce
The results of the present study indicated that SNI rats showed obvious mechanical hypersensitivity in the ipsilateral affected hindpaw, PWMT significantly decreased on the second day and reached to the peak at approximately 2 weeks after the surgery. When the SNI rats treated with SDF for 14 days, mechanical hypersensitivity of SNI rats was significantly attenuated in a dose-dependent manner. Moreover, SDF had no influence on the autonomic nerve and motor nerve of normal rats by the locomotor activity test and the rotarod test, respectively. These data suggested that SDF was the main active ingredients of SD and had potential analgesic effect on neuropathic pain. Thus, we further examined the underlying analgesic mechanism of SDF on neuropathic pain. It is well known that members of FGFR family have multiple critical roles during the formation of the CNS from the stage of neural
Fig. 7. Effects of SDF on p-CREB expression of the spinal dorsal cord in SNI rats by immunofluorescent histochemisty staining and western blotting. (A) p-CREB staining in the ipsilateral spinal cord in normal, sham, SNI and SNI treated with SDF (low, 100 mg/kg; medium, 200 mg/kg and 400 mg/kg) groups. (B) Bar histograms show the statistical analysis results of p-CREB staining in laminae I and II of the ipsilateral spinal cord of the above-mentioned groups. (C) The binds of p-CREB expression in the spinal dorsal cord of the above-mentioned groups group rats. Bar histograms show p-CREB band intensity, as a ratio over p-CREB and normalized over GAPDH. ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to SNI rats; ++ P < 0.01, +++ P < 0.001, compared to sham rats. Scale bar, 20 μm.
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induction to the stage of terminal differentiation (Ford-Perriss et al. 2001). There are four FGFR genes, FGFR-1–4, and, within these, alternative splicing creates receptor isoforms with distinct specificities for particular FGFs. In normal CNS, astrocytes are active and carry out various functions. It could support, nourish neurons and regulate the external chemical environment of neurons. However, once astrocytes are activated following peripheral inflammation or nerve injury, the expression of GFAP, as astrocytic markers, was remarkably increased in the spinal cord (Gao and Ji 2010; Johansen et al. 2001; Chen et al. 2012). Pringle et al. (2003) reported that mice with a targeted deletion in the FGFR3 locus strongly up-regulate GFAP in gray matter (protoplasmic) astrocytes, implying that signaling through FGFR3 normally represses GFAP expression in vivo. Thus, to explore the possible relationship of SDF on FGFR3 and GFAP in the spinal dorsal cord of SNI rats, we performed western blotting and/or immunohistochemical staining analysis of FGFR3 and GFAP. Western blotting results showed that FGFR3 and GFAP expression was significantly increased in the spinal dorsal horn of SNI rats. Collectively, sharply increased numbers of GFAP positive astrocytes were activated in the spinal dorsal horn of SNI rats, too. After treating with SDF for 14 days, FGFR3 and GFAP expression were dose-dependently reduced in the spinal dorsal horn of SNI rats compared to vehicle treatment. These data implied a possibility that SDF contributes to FGFR3 through repressing GFAP expression to attenuate neuropathic pain. Previous many studies have provided compelling evidence that neuropathic pain pathogenesis is not only simply confined to the changes of the activity of neuronal systems, but also involves interaction among neurons, inflammatory immune and immune-like glial cells, as well as a raft of immune cell-derived inflammatory cytokines and chemokines (Austin et al. 2010). Once activated, astrocytes lead to pro-inflammatory responses with pathological effects (Watkins et al. 2003). It was well-characterized that damage to peripheral nerves, DRG and spinal cord was associated with a rapid immune response characterized by endogenous TNF-α and IL-1β , including their proteins, mRNA and receptors (Zelenka et al. 2005; Ma et al. 1998). On the contrary, IL-10 was an anti-inflammatory factor, whose action was predominantly through suppression of pro-inflammatory cytokines at both the injury site and in the spinal cord. In our study, we found that in the spinal dorsal cord of SNI rats, levels of TNF-α and IL-1β were up-regulated and that of IL-10 was decreased as compared to healthy rats. Intragastric administration of SDF for 14 days significantly reduced TNF-α and IL-1β levels and increased level of IL-10 in a dose-dependent manner in SNI rats as compared to vehicle treatment. These results indicated SDF has a strong in vivo antiinflammatory activity via up-regulating expression of IL-10 and suppressing expression of TNF-α and IL-1β to anti-neuropathic pain. On the other hand, interestingly, NO, a mediator, is released from astrocytes which are activated (Liu et al. 2000). NO is a free radical with signaling functions in the CNS and is generated endogenously from l-arginine by NOS (Garthwaite and Boulton 1995). NO participated in nociceptive signaling after inflammation, neuropathy, and trauma (Omote et al. 2001). Increased NO level and up-regulation of NOS production in the spinal cord could lead to hyperalgesia and allodynia in rats (Tao and Johns 2002). Moreover, NO could be involved in the structural plasticity of the granule cell layer in the dentate gyrus by regulating the expression of p-CREB in the hippocampus (Park et al. 2004). Thus, NO may magnify the signal of neuropathic pain by regulating the expression of p-CREB after nerve injury. Lots of evidence suggested that p-CREB plays a vital role in developing and maintaining the chronic neuropathy pain, and participates in the peripheral and central sensitization of nociceptive neurons (Miletic et al. 2002; Ma et al. 2003). In the process of neuropathic pain, pCREB can bind to specific DNA consensus sequences, including the cyclic AMP (cAMP) response element, and can regulate the expression of immediate-early genes including c-jun (Lamph et al. 1990) and c-fos (Sheng et al. 1991), and some late-effector genes (Tsukada et al.
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1987; Watson and Latchman 1995; Messersmith et al. 1998). Some studies have reported that p-CREB expression was increased in the spinal dorsal horn following multiple pain models (Hoeger-Bement and Sluka 2003; Wang et al. 2006). To explore the possible relationship of SDF on NO and p-CREB in the spinal dorsal cord of SNI rats, we performed western blot analysis and immunohistochemical analysis of p-CREB. We observed a significant elevation of NO, and NOS levels in the spinal dorsal cord of SNI rats. These abnormalities could be alleviated by SDF treatment via a dose-dependent manner. Moreover, the present results showed that p-CREB immunopositive neurons were densely distributed in the superficial dorsal horn of SNI rats. Besides, western blotting results showed that p-CREB expression was significantly increased in the spinal dorsal horn of SNI rats. After 14 days treatment, SDF could significantly reduce the numbers of pCREB positive profiles in the spinal dorsal horn compared to SNI rats. These findings supported the possibility that SDF represses the signal amplification of pro-inflammatory cytokines through inhibiting NO/p-CREB pathway in neuropathic pain. Conclusion The present study suggests that SDF has antinociceptive effect against SNI model of neuropathic pain that might partly be related to inhibit the releasing of pro-inflammatory cytokines from astrocytes by FGFR3/GFAP and NO/GFAP pathways in the spinal dorsal horn. Furthermore, it is likely that SDF could also repress the signal amplification of pro-inflammatory cytokines by inhibiting NO/p-CREB pathway in neuropathic pain. Conflict of interest The authors declare that there is no conflict of interest regarding the publication of this article. Acknowledgments This work was supported by the National Key Technology R&D Program in 12th Five Year Plan of China (No. 2012BAI27B06), Hubei Province Natural Science Foundation of China (No. 2013CFA 013) and China Postdoctoral Science Foundation (No. 2014M560760). References Attal, N., Cruccu, G., Haanpaa, M., Hansson, P., Jensen, T.S., Nurmikko, T., Sampaio, C., Sindrup, S., Wiffen, P., 2006. EFNS guidelines on pharmacological treatment of neuropathicpain. Eur. J. Neurol. 13, 1153–1169. Austin, P.J., Moalem-Taylor, G., 2010. The neuro-immune balance in neuropathic pain: involvement of inflammatory immune cells, immune-like glial cells and cytokines. J. Neuroimmunol. 229, 26–50. Burakgazi, A.Z., Messersmith, W., Vaidya, D., Hauer, P., Hoke, A., Polydefkis, M., 2011. Longitudinal assessment of oxaliplatin-induced neuropathy. Neurology 77, 980– 986. Chen, F.F., Xiong, H., Wang, J.X., Ding, X., Shu, G.W., Mei, Z.N., 2013. Antidiabetic effect of total flavonoids from Sanguis draxonis in type 2 diabetic rats. J. Ethnopharmacol. 149, 729–736. Chen, F.L., Dong, Y.L., Zhang, Z.J., Cao, D.L., Xu, J., Hui, J., Zhu, L., Gao, Y.J., 2012. Activation of astrocytes in the anterior cingulate cortex contributes to the affective component of pain in an inflammatory pain model. Brain Res. Bull. 87, 60–66. Decosterd, I., Woolf, C.J., 2000. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 87, 149–158. Dworkin, R.H., O’Connor, A.B., Backonja, M., Farrar, J.T., Finnerup, N.B., Jensen, T.S., Kalso, E.A., Loeser, J.D., Miaskowski, C., Nurmikko, T.J., Portenoy, R.K., Rice, A.S., Stacey, B.R., Treede, R.D., Turk, D.C., Wallace, M.S., Wallacem, M.S., 2007. Pharmacologic management of neuropathic pain: evidence-based recommendations. Pain 132, 237–251. Fan, L.L., Tu, P.F., He, J.X., Chen, H.B., Cai, S.Q., 2008. Microscopical study of original plant of Chinese drug “Dragon’s Blood” Dracaena cochinchinensis and distribution and constituents detection of its resin. China J. Chin. Mater. Med. 10, 003. Ford-Perriss, M., Abud, H., Murphy, M., 2001. Fibroblast growth factors in the developing central nervous system. Clin. Exp. Pharmacol. Physiol. 28, 493–503. Gao, Y.J., Ji, R.R., 2010. Targeting astrocyte signaling for chronic pain. Neurotherapeutics 7, 482–493. Garthwaite, J., Boulton, C.L., 1995. Nitric oxide signaling in the central nervous system. Annu. Rev. Pathol. 57, 683–706.
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