Analgesic effect of coumarins from Radix angelicae pubescentis is mediated by inflammatory factors and TRPV1 in a spared nerve injury model of neuropathic pain

Author’s Accepted Manuscript Analgesic effect of coumarins from Radix angelicae pubescentis is mediated by inflammatory factors and TRPV1 in a spared nerve injury model of neuropathic pain Ruili Li, Chao Zhao, Minna Yao, Ying Song, Yin Wu, Aidong Wen www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(16)32154-7 http://dx.doi.org/10.1016/j.jep.2016.11.046 JEP10590

To appear in: Journal of Ethnopharmacology Received date: 22 June 2016 Revised date: 25 November 2016 Accepted date: 29 November 2016 Cite this article as: Ruili Li, Chao Zhao, Minna Yao, Ying Song, Yin Wu and Aidong Wen, Analgesic effect of coumarins from Radix angelicae pubescentis is mediated by inflammatory factors and TRPV1 in a spared nerve injury model of neuropathic pain, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.11.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Analgesic effect of coumarins from Radix angelicae pubescentis is mediated by inflammatory factors and TRPV1 in a spared nerve injury model of neuropathic pain ＃

Ruili Li11, Chao Zhao 1, Minna Yao1, Ying Song2, Yin Wu*1, Aidong Wen*1 1

Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Changle West Street 15,

Xi'an, Shaanxi 710032, China 2

Department of Psychiatry, Johns Hopkins University School of medicine, Wolfe Street, Baltimore,

MD, 21287, USA *

Corresponding authors: Tel. /fax: +86 29 84775475-8211. [email protected]

Abstract Ethnopharmacological relevance: Coumarins from Radix angelicae pubescentis (CRAP) are a major active component that are isolated from dried roots of Angelica biserrata Yuan et Shan, which has been used clinically to cure headaches for a long period of time, and it is an effective treatment for pain. The aim of the present study was to investigate the analgesic effect of CRAP on a spared nerve injury (SNI) model of neuropathy. Materials and methods: Antinociceptive effects of CRAP were assessed in Sprague-Dawley male rats using a spared nerve injury model of neuropathic pain. Inflammatory factors were determined by Enzyme-linked immunosorbent assay (ELISA). Transient receptor potential cation channel 1 (TRPV1) and Phosphorylated extracellular regulated protein kinases (pERK) were detected by Immunofluorescence and Western blotting, respectively. Results: The high performance liquid chromatography (HPLC) analysis showed the presence of osthole and columbianadin in Radix angelicae pubescentis. CRAP induced the dose-dependent effect of on attenuating the development of mechanical hypersensitivity. Molecular profiling revealed that CRAP reduced the levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) and significantly attenuated the expression of TRPV1 and pERK in damaged DRG neurons. Conclusion: This results demonstrate that CRAP possess remarkable antinociceptive activities which may be due to osthole and columbianadin at least in part, supporting the folkloric usage of the plant to treat various pain diseases.

Graphical abstract Post nerve 0

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7 14 ＊ S N I 1

injury （Days）

3 10

Drug admini stration

Beh

SNI:

avio r

test These authors contributed equally to this work.

spared

Fixation for IF Sacrificed for WB & ELISA

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nerve injury IF: Immunofluorescence WB: Western blotting ELISA: Enzyme-linked immunosorbent assay Abbreviations: CRAP, Coumarins from Radix angelicae pubescentis; SNI, spared nerve injury; DRG, dorsal root ganglion; CRAP, coumarins from Radix angelicae pubescentis; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; TRPV1, Transient receptor potential cation channel 1; pERK, Phosphorylated extracellular regulated protein kinases; PKC, protein kinase C; MAPKs, mitogen-activated protein kinases; LOD, limit of detection; LOQ, limit of quantification; ELISA, Enzyme-linked immunosorbent assay; RSD, relative standard deviations. Keywords: Radix angelicae pubescentis; Coumarin; Neuropathic pain; TRPV1; pERK

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Introduction Primary nervous system damage or neurological dysfunction may cause neuropathic pain, which

is a devastating neurological disease that seriously affects patients’ quality of life (Costigan et al., 2009; Treede et al., 2008; Dworkin et al., 2010). Current clinical medications for neuropathic pain relief primarily include antiepileptics, opioid analgesics, antidepressants, and topical lidocaine (Xu et al., 2012; Dworkin et al., 2007; Rice and Hill, 2006). The treatment options for neuropathic pain syndromes are not entirely effective and produce a myriad of side effects, including drug addiction, constipation, orthostatic hypotension, misuse, somnolence and hyperalgesia (Stacey and Swift, 2006; Dharmshaktu et al., 2012; Chou et al., 2009). Therefore, it is necessary to explore new pharmacological tools for the treatment of neuropathic pain. Radix angelicae pubescentis isolated from the dried roots of Angelica biserrata Yuan et Shan belong to the Umbelliferae family. Radix angelicae pubescentis was initially described in Shennong Ben Cao Jing as a traditional Chinese medicine to cure rheumatism via the elimination of inflammation and the alleviation of pain with a long history (Li et al., 2015). Radix angelicae pubescentis is used clinically in China to cure headache caused by cold weather, rheumatism and lassitude (Chang et al., 2014). Previous studies revealed that the ethanol extracts of Radix angelicae pubescentis possess potent analgesic activity. These extracts extend the incubation period of mouse writhing induced by acetic acid and prolong the tail-curl latency in mice tail immersion tests compared to aspirin (Li et al., 2013). Courmarin, which is the main active ingredient of Radix angelicae pubescentis, has been proved to be effective on pain (Park et al., 2013). CRAP are primarily composed of osthole, columbianadin, columbianedin, columbianetin, columbianetin acetate, xanthotoxin and bergapten. However, whether CRAP exerts protective effects and alleviates neuropathic pain is not known. Pain is classically transmitted from sensory nerve endings to the dorsal root ganglion (DRG), spinal cord and cerebrum (Chizh and Illes, 2001; Glombiewski et al., 2010). Neuropathic pain may arise from discharge at the site of axonal injury and spontaneous activity in DRG neurons (Thakor et al., 2009; Ma et l., 2003; Wu et al., 2001). Numerous inflammatory cytokines initially gather in the injured tissue, lower activation thresholds in nociceptors produces neuropathic pain (Xu et al., 2012). Numerous studies demonstrated that inflammation and the immune response play important roles in neuropathic pain. Pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6, exhibit temporal

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up-regulation, which leads to a series of changes in DRG neurons that contribute to the development of neuropathic pain after nerve injury (Cao et al., 2015; Hwang et al., 2005). TRPV1 is a member of the TRP family proteins, and it is widely distributed in DRG neurons. TRPV1 is involved in pain transduction, transmission, perception and modulation (Hwang et al., 2005; Jara-Oseguera et al., 2008; Starowicz et al., 2008). Activated TRPV1 facilitates cation flux from the extracellular to intracellular space, which leads to the release of substance P and calcitonin-gene related peptide (CGRP) and pain generation (Szallasi and Sheta, 2012). TRPV1 expression is down-regulated in damaged DRGs after peripheral nerve injury, but it is up-regulated in the undamaged population (Fukuoka et al., 2002; Hudson et al., 2001). Numerous endogenous and exogenous signals regulate TRPV1 activity and promote pain generation or sensitization, such as G protein-coupled receptors, tyrosine kinase receptors, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs), which are associated with TRPV1 sensitization (Ferrari et al., 2014; Ji et al., 2002; Palazzo et al., 2012). ERK modulation of TRPV1 activity plays a critical role in pain perception, and it has been widely studied in various pain models (Jin et al., 2003; Svensson et al., 2003). ERK phosphorylation is necessary to maintain the cytokine-mediated TRPV1 sensitization in sensory neurons (Hensellek et al., 2007). It is reported that coumarin derivative specifically activate the nociceptor TRPV1 and reverse the inflammatory pain in mice through channel desensitization (Wei et al., 2016). However, how CRAP act on pain control is still not clear. This study examined the relationships between CRAP and pain processing in a SNI model of neuropathic pain. We also measured the expression of kinases that influence TRPV1 sensitization and correlated changes in TNF-α, IL-1β and IL-6 expression during the development of allodynia. These studies provide a mechanistic framework for further studies of the use of CRAP as an effective treatment for neuropathic pain. 2.

Materials and Methods

2.1. Drugs and agent Radix angelicae pubescentis was purchased from the Xijing hospital pharmacy. CRAP was prepared in our laboratory. Briefly, Radix angelicae pubescentis was ground into a powder and 10g powder was extracted with 500 mL ethanol twice for 2 h under ultrasonic oscillation at room temperature (25 °C). The ethanol was retrieved, and the extract was concentrated in a Rotary evaporation instrument for approximately 0.5 h at 60°C and transferred to an evaporating dish. The dish was placed in a water bath at 60°C until the extract reached a density of 1.10 at 20 °C (Li et al., 2013; Chen et al., 2015). For qualitative determination of CRAP, a LC-10A HPLC instrument (Shimadzu, Japan), equipped with a SPD-10A detector was used. The UV spectra were recorded between 190 and 490 nm for peak characterization, and the detection wavelength was set at 322 nm. A Diamond C18 (4.6 mm×250 mm, 5 μm) was used with the column temperature held at 25 °C. Elution was carried out at a flow rate of 1.0 mL/min with mobile phases consisting of ethanol-acetonitrile-0.025 mol/L Phosphoric acid (43:20:37). The mobile phase was filtered through a Millipore 0.45 μm filter and degassed prior to use. The peaks were detected at 322 nm and osthole and columbianadin were detected by comparing with the chemical marks, which were identified with MS, 1H NMR, and 13C NMR. Analytical method’s linearity, limit of detection and quantification (LOD and LOQ) and inter-day/intra-day precision were validated following the ICH guidelines (ICH 1996). Recovery was used to evaluate the accuracy of the method. Two standard references, namely, osthole (1, >98%, CAS: 484-12-8) and columbianadin (2, >98%, CAS: 5058-13-9) were purchased from Shaanxi Pioneer Biotech Co.,Ltd. (Xi’an, China).The

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antibodies against TRPV1 and pERK were obtained from Signaling Technology (Beverly, MA). TNF-α, IL-1β and IL-6 ELISA kits were purchased from R&D Systems (Minneapolis, MN). 2.2. Animals and Experimental Design Sprague-Dawley male rats, 180-220 g, were supplied by the Experimental Animal Center, Fourth Military Medical University. The Animal Care and Use Committee of the Fourth Military Medical University reviewed and approved the use of animals (approval number: XJYYLL-2015612). This experiment was conducted in accordance with the guidelines issued by the Chinese Food and Drug Administration (cFDA). Rats were raised under controlled room humidity (45-75%) and temperature (22 ± 2 °C) with 12/12 h light/dark cycles and provided food and water ad libitum. Rats were acclimatized to the laboratory conditions for 1 week prior to experiments. All efforts were made to minimize the number of animals used and animal suffering. The experiment was designed as follows. The analgesic effects of CRAP in neuropathic pain were investigated. Forty-two rats were divided into 7 groups (n = 6): sham-operated group, sham-operated treated with CRAP (20 mg/kg) group, SNI model treated with CRAP (0, 5, 10, or 20 mg/kg) and morphine (3 mg/kg) groups. CRAP was administered intra-gastrically after surgery from post-operative day (POD) 0 to POD 14. The sham group and SNI model group received an equal volume of vehicle. Behavioral experiments were performed once daily on POD 0, 1, 3, 7, 10 and 14. The roles of TNF-α, IL-1β and IL-6 in the analgesic effects of CRAP were assessed on POD 14. Eighteen rats were divided into 3 groups (n = 6): the sham-operated group, SNI model group and SNI model treated with CRAP (20 mg/kg) group. Rats were used for enzyme-linked immunosorbent assay (ELISA) of SNI injury each day. The role of TRPV1 in the process of neuropathic pain was investigated after CRAP treatment on POD 14. Thirty-six rats were divided into 3 groups (n = 12): sham-operated group, SNI model group and SNI model treated with CRAP (20 mg/kg) group. Six rats in each group were used for immunofluorescence, and six rats were used for western blotting. ERK phosphorylation level was detected using western blotting on POD 14. 2.3. SNI induction in rats We used the SNI model of neuropathic pain (Decosterd and Woolf, 2000). Briefly, rats were anesthetized with 1% pentobarbital sodium (5 ml/kg) intraperitoneally (i.p.), and the skin on the lateral surface of the thigh was disinfected with iodine under aseptic conditions. The skin was incised and subcutaneous tissue and muscle were blunt separated to expose the sciatic nerve and its three terminal branches: the sural, common peroneal and tibial nerves. The tibial and common peroneal nerves were tightly ligated using 5.0 silk, and 2-3 mm of the nerve were cut distal to the ligation. The sural nerve was left intact, and the wound was closed. A small amount of penicillin sodium powder was applied to the incision. 2.4. Mechanical Hypersensitivity Paw withdrawal in response to mechanical stimuli was assessed using calibrated von Frey filaments (Stoelting, Kiel, WI, USA). Rats were placed on an elevated mesh grid that completely exposed the middle of the hind paw. Animals were habituated to the testing environment for at least 0.5 h prior to each testing session. The stiffness values of the von Frey filaments were 2, 4, 6, 8, 10, 15 and 26 g. Each filament was applied to the hind paw in an order of increasing stiffness for 5 s. Rapid pulling back, biting, or shaking of the hind limb within 5 s of the application of the von Frey filament to the hind paw was taken as a positive sign of withdrawal. The interval between trials was at least 5 min. The same hind limb was stimulated 10 times by a single von Frey filament for each trial before

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stimulation by the next larger filament. The minimum value that resulted in at least six responses to ten stimulations was recorded as the paw withdrawal threshold (Tang et al., 2015). The formula for calculating the percent change was 100－100×(SSNI+CRAP－SSNI+Veh)／(SSham+Veh－SSNI+Veh), with S as the stiffness of the von Frey filaments. 2.5. Enzyme-linked immunosorbent assay Rats were decapitated under anesthesia with an over-dose of 1% pentobarbital sodium. The ipsilateral L5 DRG was rapidly harvested and homogenized in PBS. Homogenates were centrifuged at 4 °C for 15 min at 900×g, and the supernatants were used to measure the concentrations of TNF-α, IL-1β and IL-6 using corresponding ELISA kits. 2.6. Immunofluorescence The rats were anesthetized with 1% pentobarbital sodium and perfused through the aorta with 200 mL normal saline, followed by 400 mL 4% paraformaldehyde. The ipsilateral L5 DRG was removed, post-fixed in 4% paraformaldehyde for 2 h, and cryoprotected in a 30% sucrose solution. The DRG was cut into 20 μm-thick serial sections using a cryostat (Leica CM1950) and mounted on 3-aminopropyl-triethoxysilane-coated glass slides. DRG sections were stored at -20 °C. Immunofluorescence was performed as follows. DRG sections were air-dried for 4-6 h and fixed with 4% paraformaldehyde for 0.5 h. Sections were washed in PBS, incubated with 3% bovine serum albumin containing 0.4% Triton X-100 for 1 h, and incubated with a mixture of TRPV1 (1:200) and NeuN (1:1000) antibodies overnight at room temperature. Sections were washed with PBS and incubated with a mixture of appropriate fluorochrome-conjugated secondary antibodies at room temperature for 3 h in the dark. Sections were washed 3 times for 5 min with PBS and cover slipped using mounting liquid. Stained sections were visualized, and photographs were obtained under a fluorescent microscope (Leica Microsystems GmbH, Mannheim, Germany). 2.7. Western blot analysis Western blot was performed as described in our previous report (Li et al., 2014). Briefly, rats were decapitated under anesthesia with an overdose of 1% pentobarbital sodium. The ipsilateral L5 DRG was removed, quickly frozen in liquid nitrogen and stored at -80 °C. Frozen tissues were incubated in tissue-lysing buffer containing protease inhibitors on ice for 30 min. Protein concentration was determined using a BCA protein assay. Each sample containing 30 μg of protein was mixed with a one-fifth volume of 5× loading buffer and heated to 100 °C for 5 min. Protein samples and rainbow-colored protein molecular markers were separated using 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.5% Tween-20 (TBST) for 30 min before probing with primary antibodies containing 5% BSA at 4 °C overnight. Membranes were washed with TBST for 30 min and probed with appropriate secondary antibodies conjugated with horseradish peroxidase for 1 h. Immunoreactive bands were detected using enhance chemiluminescence (ECL) detection reagents, and the intensity of protein bands was quantified using densitometry in Image ProPlus 6.0 software. 2.8. Statistical analysis Data are presented as the means ± S.E.M. of triplicate determinations, except when the results of blots are shown, in which case a representative experiment is depicted in each figure. Comparisons between multiple groups were performed using one-way ANOVA or two-way ANOVA with Bonferroni correction. Statistical significance was indicated when p < 0.05. 3.

Results

3.1 HPLC analysis of CRAP

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In the present study, firstly we carried out the qualitative and quantitative analysis of CRAP. With RP-HPLC, peaks of CRAP were compared with chemical marks, osthole (1, Rt: 19.812 min), and columbianadin (2, Rt: 23.476 min) were proved as the main components of CRAP (Fig.1). Quantitative results of two constituents and method validation were seen in Table 1-3. Linearity and calibration curves. Linearity of four compounds calibration curves was established by calculating the slope, intercepts and r2 coefficient. Two compounds were demonstrated to have good linear regression with high correlation coefficient values between peak area (Y) and amount (X, μg). All the analytes showed good linearity (r2>0.999) in concentration ranges (Table 1). LOD and LOQ were calculated as 3.3 σ/S and 10 σ/S respectively, being σ the response standard deviation, and S the slope of each marker. Precision, repeatability and stability. Intra- and inter-day variability test was assessed by repetitive injections of the same sample solution for six times in 1 d. Variations were expressed by the relative standard deviations (RSD) (Table 2), confirming the precision of the proposed method. Repeatability was determined by analyzing six independently prepared samples of CRAP using the same method. Stability was evaluated by analysis of six injections of the same sample solution every 3 h at 25 °C. These data were shown in Table 2. The results indicated that the sample remained stable for 12 h. Recovery and accuracy. The recovery test was carried out by spiking certain known quantity of the two references with pulverized sample (0.25 g) of CRAP and six samples were prepared for the test simultaneously. It is an acceptable result for recovery analysis (Table 3). Three concentrations of pre-analyzed sample solutions were spiked with known quantities of the standards and injected in triplicate to perform recovery studies. The percentage recovery for two compounds were between 98.10 and 100.71% (RSD < 2 %, n = 3), confirming the accuracy of the proposed method. The valid method was employed to determine the four ingredients, and the contents of ingredients were detected: 1 (0.51%), 2 (0.09%). 3.2 CRAP attenuated SNI-induced mechanical hypersensitivity in a dose-dependent manner The acceptor/ion channels in damaged DRG after SNI alter neuronal electrical activity to noxious stimuli, such as mechanical, heat and cold, which leads to the release of excitatory neurotransmitters and neuronal hyper-excitability in the spinal dorsal horn, which is called central sensitization. We established SNI as a neuropathic pain model to evaluate the effect of CRAP on preventing mechanical hypersensitivity. Fig. 2A shows that intra-gastric administration of 20 mg/kg CRAP did not influence basal thresholds in the sham-operated group. SNI injury resulted in prominent mechanical hypersensitivity (p < 0.05). However, CRAP treatment altered paw withdrawal thresholds (PWTs) of ipsilateral hind paws from POD 3 to POD 14 compared to the SNI model group, similar to the effects of morphine (3 mg/kg) (p < 0.05). We also found significant differences in PWTs in the groups treated with different CRAP doses, which suggests that CRAP inhibited mechanical hypersensitivity in a dose-dependent manner. We calculated the slope factor and ED50 of SNI-induced mechanical hypersensitivity from the dose-response curve (Fig. 2B). The ED50 of CRAP for SNI-induced mechanical hypersensitivity was 10.28 mg/kg, and the slope factor was 1.554, which suggest that the dose selection was reasonable in our experiment. 3.3 CRAP reduced SNI-induced up-regulation of pro-inflammatory cytokines in damaged DRG Inflammation and the immune response play important roles in neuropathic pain. We examined the effect of CRAP on the expression of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6, using ELISA. Fig. 3 shows that the levels of TNF-α (Fig. 3A), IL-1β (Fig. 3B) and IL-6 (Fig. 3C) were lower in the sham-operated group than the SNI model group on POD 14 (p < 0.05). We observed a

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significant increase in the protein expression of TNF-α, IL-1β and IL-6 in the SNI model group compared to the sham-operated group. Treatment with CRAP attenuated the expression of TNF-α, IL-1β and IL-6 on POD 14 (p<0.05). These results suggest that intra-gastric CRAP effectively inhibited SNL-induced up-regulation of pro-inflammatory cytokines. 3.4 CRAP increased TRPV1 expression in damaged DRG neurons Localization of TRPV1 in rat ipsilateral DRG was determined using immunofluorescence on POD 14. Immunofluorescence-labeled TRPV1 and mature neuronal marker NeuN antibodies were used. Colocalized NeuN and TRPV1 was merged. Fig. 4A shows that the population of TRPV1-positive neurons in the SNI model group was lower than the sham-operated group. CRAP treatment significantly increased TRPV1 expression in damaged DRG neurons on POD 14, which suggests that TRPV1 is involved in the process of neuropathic pain after CRAP administration. Western blotting of TRPV1 levels in the ipsilateral DRG of SNI rats was consistent with the immunofluorescence results (Fig. 4B and C). CRAP treatment increased the level of TRPV1 in ipsilateral DRG compared to SNI rats (p < 0.05). 3.5 CRAP decreased ERK phosphorylation in damaged DRG ERK is an important molecule that is closely associated with pain. ERK expression was investigated using western blot analysis. Our results indicated that pERK expression was increased in the SNI model group compared to the sham-operated group, and CRAP treatment reversed pERK activation compared to the SNI model group (p < 0.05) (Fig. 5). 4.

Discussion This study reports the anti-nociceptive effects and molecular mechanisms of action of CRAP.

Behavioral testing demonstrated that CRAP administration alleviated mechanical allodynia in neuropathic rats. CRAP also suppressed the production of pro-inflammatory cytokines. We also demonstrated that the increased abundance of TRPV1 in damaged DRG neurons was involved in the anti-nociceptive effect of CRAP. CRAP significantly attenuated pERK expression, which is a known downstream effector in the MAPK pathway. Taken together, our results indicate that the analgesic effect of CRAP is likely mediated wholly or in a large part by the activation of TRPV1 after SNI injury. Accordingly, we used morphine as positive control. This finding may provide a mechanistic framework for the further investigation of CRAP as an effective treatment for neuropathic pain. Neuropathic pain is characterized by the spontaneous discharge of primary sensory neurons and continuously improving transfer efficiency of synapses in the spinal dorsal horn after peripheral nerve injury. Hyperalgesia is a notable feature of neuropathic pain. DRG of primary sensory neurons are associated with a rapid immune response that is characterized by the production of endogenous cytokines, such as TNF-α, IL-1β and IL-6 and their receptors (Schafers et al., 2003), ion channels and receptors such as TRP channels, P2X and P3Y receptors, (Hudson et al., 2001; Song et al., 2015) and the molecules (such as PKC and MAPKs) that regulate these channels (Ferrari et al., 2014; Ji et al., 2002; Palazzo et al., 2012). TRPV1 has been a target molecule of important therapeutic approaches. Experimental results demonstrated that CRAP significantly increased the TRPV1 expression in DRG neurons. The present study used a well-established neuropathic pain model, SNI, to investigate the anti-nociceptive effects of CRAP. Behavioral testing demonstrated that CRAP administration alleviated SNI-induced mechanical hypersensitivity (Fig. 2 A). CRAP markedly inhibited the protein expression of TNF-α, IL-1β and IL-6 in ipsilateral L5 DRG (Fig. 3). We also demonstrated that CRAP significantly increased the TRPV1 expression in damaged DRG neurons (Fig. 4) and significantly

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decreased pERK expression (Fig. 5). These results provide a theoretical basis for the clinical application of CRAP. We evaluated pain behavior using the Von Frey test to measure the development of neuropathic pain. The results demonstrated that neuropathic pain began on POD 3 and was present until POD 14. We detected the pro-inflammatory proteins, TRPV1 and pERK, on POD 14. TRPV1 was located in primary afferent nociceptive neurons in DRG, which play an important role in neuropathic pain. TRPV1 mediates the development of neuropathic pain regardless of its expression level (Patapoutian et al., 2009). Neuropathic pain is associated with a series of inflammatory mediators, including cytokines (Ren and Dubner, 2010). TNF-α is an important pro-inflammatory cytokine that may contribute to TRPV1 activity. TNF-α pretreatment may modulate the sensitivity of the TRPV1 receptor to endogenous and exogenous ligands and result in increased nociceptive signal transmission (Khan et al., 2008; Spicarova and Palecek, 2009). IL-1β was up-regulated in our SNI model, which suggests its involvement in the development and maintenance of neuropathic pain. IL-1β contributes to TRPV1 receptor sensitization via a peripheral mechanism (Obreja et al., 2002). IL-6 is also elevated because of TRPV1 activity after the development of the pain phenotype (Terenzi et al., 2013). Sensitization of TRPV1 receptors by pro-inflammatory agents involves ERK-dependent phosphorylation. The activation of ERK plays an important role in neuropathic pain. ERK is phosphorylated when inflammatory mediators stimulate sensory neurons (Galan et al., 2003; Ji, 2004). We observed that TRPV1 expression was significantly decreased in damaged L5 DRG, which indicated an increase in TRPV1 in undamaged DRG (not mentioned in our paper) in an SNI model of neuropathic pain. The present findings are consistent with previous results (Fukuoka et al., 2003; Hudson et al., 2001). The increase in TRPV1 is mediated by ERK phosphorylation during pain (Sun et al., 2012). This study found increased ERK phosphorylation in neuropathic pain. These results suggest that pERK expression is linked to TRPV1. 5.

Conclusions CRAP significantly prevented neuropathic pain and attenuated the development of mechanical

hypersensitivity induced by SNI. This study suggests that the anti-nociceptive activities of CRAP are associated with pro-inflammatory cytokines (TNF-α, IL-1β and IL-6), TRPV1 and pERK in peripheral nervous pain systems. Therefore, CRAP is a promising candidate as a therapeutic anti-nociceptive agent. Conflict of interest The authors declare that there are no conflicts of interest in this paper. Acknowledgments This work was supported by a grant from the National New Drug “R&D” project (No. 2011ZXJ09302). The authors are grateful for the proofreading of this manuscript by Mr. Zongtao Lin in the Department of Pharmaceutical Sciences, University of Tennessee Health Science Center and Mr. Bo Wang in the Department of Developmental Neurobiology, St. Jude Children’s Research Hospital. References Cao, F.L., Xu, M., Wang, Y., Gong, K.R., Zhang, J.T., 2015. Tanshinone IIA attenuates neuropathic pain via inhibiting glial activation and immune response. Pharmacol Biochem Behav 128, 1-7. Chang, Y.X., Wang, C.P., Li, J., Bai, Y., Luo, Q., He, J., Lu, B., Wang, T., Zhang, B.L., Gao, X.M., 2014. LC-MS/MS determination and pharmacokinetic study of columbianadin in rat plasma after intravenous administration of pure columbianadin. Chem Cent J 8, 64. Chen, F.F., Huo, F.Q., Xiong, H., Wan, Q., Zheng, Y.N., Du, W.J., Mei, Z.N., 2015. Analgesic effect of

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total flavonoids from Sanguis draxonis on spared nerve injury rat model of neuropathic pain. Phytomedicine 22, 1125-1132. Chizh, B.A., Illes, P., 2001. P2X receptors and nociception. Pharmacol Rev 53, 553-568. Chou, R., Fanciullo, G.J., Fine, P.G., Adler, J.A., Ballantyne, J.C., Davies, P., Donovan, M.I., Fishnain, D.A., Foley, K.M., Fudin, J., Gison, A.M., Kelter, A., Mauskop, A., O'Connor, P.G., Passik, S.D., Pasternak, G.W., Portenoy, R.K., Rich, B.A., Roberts, R.G., Todd, K.H., Miaskowski, C., 2009. Clinical guidelines for the use of chronic opioid therapy in chronic noncancer pain. J Pain 10, 113-130. Costigan, M., Scholz, J., Woolf, C.J., 2009. Neuropathic pain: a maladaptive response of the nervous system to damage. Annu Rev Neurosci 32, 1-32. Decosterd, I., Woolf, C.J., 2000. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 87, 149-158. Dharmshaktu, P., Tayal, V., Kalra, B.S., 2012. Efficacy of antidepressants as analgesics: a review. J Clin Pharmacol 52, 6-17. Dworkin, R.H., Malone, D.C., Panarites, C.J., Armstrong, E.P., Pham, S.V., 2010. Impact of postherpetic neuralgia and painful diabetic peripheral neuropathy on health care costs. J Pain 11, 360-368. 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.C., Stacey, B.R., Treede, R.D., Turk, D.C., Wallace, M.S., 2007. Pharmacologic management of neuropathic pain: evidence-based recommendations. Pain 132, 237-251. Ferrari, L.F., Bogen, O., Levine, J.D., 2014. Second messengers mediating the expression of neuroplasticity in a model of chronic pain in the rat. J Pain 15, 312-320. Fukuoka, T., Tokunaga, A., Tachibana, T., Dai, Y., Yamanaka, H., Noguchi, K., 2002. VR1, but not P2X(3), increases in the spared L4 DRG in rats with L5 spinal nerve ligation. Pain 99, 111-120. Galan, A., Cervero, F., Laird, J.M., 2003. Extracellular signaling-regulated kinase-1 and -2 (ERK 1/2) mediate referred hyperalgesia in a murine model of visceral pain. Brain Res Mol Brain Res 116, 126-134. Glombiewski, J.A., Hartwich-Tersek, J., Rief, W., 2010. Depression in chronic back pain patients: prediction of pain intensity and pain disability in cognitive-behavioral treatment. Psychosomatics 51, 130-136. Hensellek, S., Brell, P., Schaible, H.G., Bräuer, R., Sedond von Banchet, G., 2007. The cytokine TNFalpha increases the proportion of DRG neurones expressing the TRPV1 receptor via the TNFR1 receptor and ERK activation. Mol Cell Neurosci 36, 381-391. Hudson, L.J., Bevan, S., Wotherspoon, G., Gentry, C., Fox, A., Winter, J., 2001. VR1 protein expression increases in undamaged DRG neurons after partial nerve injury. Eur J Neurosci 13, 2105-2114. Hwang, S.J., Oh, J.M., Valtschanoff, J.G., 2005. Expression of the vanilloid receptor TRPV1 in rat dorsal root ganglion neurons supports different roles of the receptor in visceral and cutaneous afferents. Brain Res 1047, 261-266. Jara-Oseguera, A., Simon, S.A., Rosenbaum, T., 2008. TRPV1: on the road to pain relief. Curr Mol Pharmacol 1, 255-269. Ji, R.R., 2004. Mitogen-activated protein kinases as potential targets for pain killers. Curr Opin Investig Drugs 5, 71-75. Ji, R.R., Samad, T.A., Jin, S.X., Schmoll, R., Woolf, C.J., 2002. p38 MAPK activation by NGF in

9

primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron 36, 57-68. Jin, S.X., Zhuang, Z.Y., Woolf, C.J., Ji, R.R., 2003. p38 mitogen-activated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. J Neurosci 23, 4017-4022. Khan, A.A., Diogenes, A., Jeske, N.A., Henry, M.A., Akopian, A., Hargreaves, K.M., 2008. Tumor necrosis factor Alpha enhances the sensitivity of rat trigeminal neurons to capsaicin. Neuroscience 155, 503-509. Li, J., Zhang, Q.H., He, J., Liu, E.W., Gao, X.M., Chang, Y.X., 2015. An Improved LC-MS/MS Method for Simultaneous Determination of the Eleven Bioactive Constituents for Quality Control of Radix Angelicae Pubescentis and Its Related Preparations. The Scientific World J, 365093. Li, R.L., Cui, B.L., Li, Y.W., Zhao, C., Jia, N., Wang, C., Wu, Y., Wen A.D., 2014. A new synthetic Cu(II) compound, [Cu3(p-3-bmb)2Cl4.(CH3OH)2]n, inhibits tumor growth in vivo and in vitro. Eur J Pharmacol 724, 77-85. Li, X., Wang, J., Gao, L., 2013. Anti-inflammatory and analgesic activity of R.A.P. (Radix Angelicae Pubescentis) ethanol extracts. Afr J Tradit Complement Altern Med 10, 422-426. Ma, C., Shu, Y., Zheng, Z., Chen, Y., Yao, H., Greenquist, K.W., White, F.A., LaMotte, R.H., 2002. Similar electrophysiological changes in axotomized and neighboring intact dorsal root ganglion neurons. J Neurophysiol 89,1588-1602. Obreja, O., Rathee, P.K., Lips, K.S., Distler, C., Kress, M., 2002. IL-1 beta potentiates heat-activated currents in rat sensory neurons: involvement of IL-1RI, tyrosine kinase, and protein kinase C. FASEB J 16, 1497-1503. Palazzo, E., Luongo, L,, de Novellis, V., Rossi, F., Marabese, I., Maione, S, 2012. Transient receptor potential vanilloid type 1 and pain development. Curr Opin Pharmacol 12, 9-17. Park, S.H., Sim, Y.B., Kang, Y.j., Kim, S.S., Kim, C.H., Kim, S.J., Lim, S.M., Suh, H.W., 2013. Antinociceptive profiles and mechanisms of orally administered coumarin in mice. Biol Pharm Bull 36, 925-930. Patapoutian, A., Tate, S., Woolf, C.J., 2009. Transient receptor potential channels: targeting pain at the source. Nat Rev Drug Discov 8, 55-68. Ren, K., Dubner, R., 2010. Interactions between the immune and nervous systems in pain. Nat Med 16, 1267-1276. Rice, A.S., Hill, R.G., 2006. New treatments for neuropathic pain. Annu Rev Med 57, 535-551. Schäfers, M., Svensson, C.I., Sommer, C., Sorkin, L.S., 2003. Tumor necrosis factor-alpha induces mechanical allodynia after spinal nerve ligation by activation of p38 MAPK in primary sensory neurons. J Neurosci 23, 2517-2521. Song, T., Wang, L., Gu, K., Yang, Y., Yang, L., Ma, P., Ma, X., Zhao, J., Yan, R., Guan, J., Wang, C., Qi, Y., Ya, J., 2015. Involvement of peripheral TRPV1 channels in the analgesic effects of thalidomide. Neurochem Int 85-86, 40-45. Spicarova, D., Palecek, J., 2009. The role of the TRPV1 endogenous agonist N-Oleoyldopamine in modulation of nociceptive signaling at the spinal cord level. J Neurophysiol 102, 234-243. Stacey, B.R., Swift, J.N., 2006. Pregabalin for neuropathic pain based on recent clinical trials. Curr Pain Headache Rep 10, 179-184. Starowicz, K., Cristino, L., Di Marzo, V., 2008. TRPV1 receptors in the central nervous system: potential for previously unforeseen therapeutic applications. Curr Pharm Des 14, 42-54.

10

Sun, S., Yin, Y., Yin, X., Cao, F., Luo, D., Zhang, T., Li, Y., Ni, L., 2012. Anti-nociceptive effects of Tanshinone IIA (TIIA) in a rat model of complete Freund's adjuvant (CFA)-induced inflammatory pain. Brain Res Bull 88, 581-588. Svensson, C.I., Hua, X.Y., Protter, A.A., Powell, H.C., Yaksh, T.L., 2012. Spinal p38 MAP kinase is necessary for NMDA-induced spinal PGE(2) release and thermal hyperalgesia. Neuroreport 14, 1153-1157. Szallasi, A., Sheta, M., 2012. Targeting TRPV1 for pain relief: limits, losers and laurels. Expert Opin Investig Drugs 21, 1351-1369. Tang, J., Zhu, C., Li, Z.H., Liu, X.Y., Sun, S.K., Zhang, T., Luo, Z.J., Zhang, H., Li, Y.W., 2015. Inhibition of the spinal astrocytic JNK/MCP-1 pathway activation correlates with the analgesic effects of tanshinone IIA sulfonate in neuropathic pain. J Neuroinflammation 12, 57. Terenzi, R., Romano, E., Manetti, M., Peruzzi, F., Nacci, F., Matucci-Cerinic, M., Guiducci, S., 2013. Neuropeptides activate TRPV1 in rheumatoid arthritis fibroblast-like synoviocytes and foster IL-6 and IL-8 production. Ann Rheum Dis 72, 1107-1109. Thakor, D.K., Lin, A., Matsuka, Y., Meyer, E.M., Ruangsri, S., Nishimura, I., Spigelman, I., 2009. Increased peripheral nerve excitability and local NaV1.8 mRNA up-regulation in painful neuropathy. Mol Pain 5, 14. Treede, R.D., Jensen, T.S., Campbell, J.N., Cruccu, G., Dostrovsky, J.O., Griffin, J.W., Hansson, P., Hughes, R., Nurmikko, T., Serra, J., 2008. Neuropathic pain: redefinition and a grading system for clinical and research purposes. Neurology 70, 1630-1635. Wei, N.N., Lv, H.N., Wu, Y., Yang, S.L., Sun, X.Y., Lai, R., Jiang, Y., Wang, K., 2016. Selective Activation of Nociceptor TRPV1 Channel and Reversal of Inflammatory Pain in Mice by a Novel Coumarin Derivative Muralatin L from Murraya alata. J BIOL CHEM 291, 640-651. Wu, G., Ringkamp, M., Hartke, T.V., Murinson, B.B., Campbell, J.N., Griffin, J.W., Meyer, R.A., 2001. Early onset of spontaneous activity in uninjured C-fiber nociceptors after injury to neighboring nerve fibers. J Neurosci 21, RC140. Xu, C., Xu, W., Xu, H., Xiong, W., Gao, Y., Li, G., Liu, S., Xie, J., Tu, G., Peng, H., Qiu, S., Liang, S., 2012. Role of puerarin in the signalling of neuropathic pain mediated by P2X3 receptor of dorsal root ganglion neurons. Brain Res Bull 87, 37-43. Xu, Y., Qiu, H.Q., Liu, H., Liu, M., Huang, Z.Y., Yang, J., Su, Y.P., Yu, C.X., 2012. Effects of koumine, an alkaloid of Gelsemium elegans Benth., on inflammatory and neuropathic pain models and possible mechanism with allopregnanolone. Pharmacol Biochem Behav 101, 504-514.

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A

B

Fig. 1. HPLC analysis of CRAP. (A) HPLC chromatogram of the sample used in the experiment; (B) HPLC chromatogram of standard reference. Peaks were detected at 322 nm (1, osthole; 2, columbianadin).

A

12

B

Fig. 2. CRAP attenuated SNI-induced mechanical hypersensitivity in a dose-dependent manner. Intra-gastric administration of 20 mg/kg CRAP did not influence basal thresholds in the sham-operated group. SNI injury resulted in prominent mechanical (A). *p < 0.05, compared to the sham-operated group. CRAP treatment influenced the paw withdrawal threshold (PWT) of ipsilateral hind paws from POD 3 to POD 14, similar to the effects of morphine (3 mg/kg), #p < 0.05, compared to the SNI + vehicle group. The log (dose) effect curves for the analgesic effects of CRAP are shown in (B), n = 12 for each group.

A

13

B

C

Fig. 3. CRAP reduced SNI-induced up-regulation of pro-inflammatory cytokines in the DRG. The expression of pro-inflammatory cytokines, including TNF-α (A), IL-1β (B) and IL-6 (C), was detected using enzyme-linked immunosorbent assays. We observed a significant increase in the protein expression of TNF-α, IL-1β and IL-6 in the SNI model group. *p < 0.05, compared to the sham-operated group. Treatment with CRAP attenuated the expression of TNF-α, IL-1β and IL-6. #p < 0.05, compared to the SNI + vehicle group.

A

14

B

Fig. 4. CRAP increased TRPV1 expression in DRG neurons. TRPV1 levels in the ipsilateral DRG of SNI rats were detected using immunofluorescence (A) and western blot (B) assays. The population of TRPV1-positive neurons in the SNI model group was lower than the sham-operated group. *p < 0.05, compared to the sham-operated group. Treatment with CRAP significantly attenuated the expression of TRPV1 in DRG neurons. CRAP treatment increased the level of TRPV1 in ipsilateral DRG. *p < 0.05, compared to the SNI rats, #p < 0.05, compared to the SNI + vehicle group.

15

Fig. 5. CRAP decreased ERK phosphorylation in DRG. The expression of pERK was increased in the SNI model group compared to the sham-operated group. *p < 0.05, compared to the sham-operated group. Treatment with CRAP reversed pERK activation. #p < 0.05, compared to the SNI + vehicle group.

Table 1. Linearity of two ingredients in CRAP (n=6) (1, osthole; 2, columbianadin) Compounds

Regression equation

r2

Linear rang

LOD

LOQ

(μg)

(ng)

(ng)

1

Y=85702X-13174

0.9997

0.37-3.81

1.16

4.15

2

Y=3951X+2705

0.9993

0.18-3.52

0.97

3.01

Table 2. Precision, repeatability and stability of two ingredients in CRAP (n=6) (1, osthole; 2, columbianadin) Compounds

Precision

LOD

Stability

Intra-day RSD (%)

Inter-day RSD (%)

RSD (%)

1

0.38

0.45

0.75

2

0.39

0.38

0.75

Table 3. Recovery and accuracy of two ingredients in CRAP (n=6) (1, osthole; 2, columbianadin) Analyte 1

2

Original

Spiked

Found

Recovery

Mean

RSD

(μg)

(μg)

(μg)

(%)

(%)

(%)

1270

400

1667

99.25

99.76

0.75

800

2075

100.63

1200

2463

99.41

70

289

98.57

99.13

1.39

140

361

100.71

210

426

98.10

220

16