{'fraction_non_alphanumeric': 0.039477914228766155, 'fraction_numerical': 0.024561177907799138, 'mean_word_length': 4.784306433618445, 'pattern_counts': {'":': 0, '<': 10, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Baicalin relieves neuropathic pain by regulating α2-adrenoceptor levels in rats following spinal nerve injury

In the present study, the ability of baicalin to relieve neuropathic pain due to spinal nerve ligation in rats was explored, and the relationship between baicalin and α2-adrenoceptors (α2-AR) was determined. The neuropathic pain model was established by ligating the L5-L6 spinal nerves in Sprague-Dawley rats. Several α2-AR antagonists were injected into the intramedullary sheath to evaluate the role of baicalin in neuropathic pain. The antagonists included nonselective α2-AR antagonist idazoxan, α2a-AR antagonist BRL 44408, α2b-AR antagonist ARC 239 and α2c-AR antagonist JP 1302. The rats were divided into an untreated control group, saline group, baicalin group and baicalin + α2-AR antagonist groups. Paw withdrawal threshold (PWT) was tested to assess the level of pain felt by the rats. The levels of α2-AR mRNA were tested by reverse transcription-quantitative PCR. Inflammatory factors, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-17 and IL-1β, were analyzed by ELISA. The histopathological changes were assessed by hematoxylin and eosin staining. Flow cytometry was used to examine the percentage of CD4+ peripheral blood mononuclear cells (PBMCs). Compared with the saline group, the PWT value increased after treating with baicalin. However, intrathecal injection of α2-AR antagonist reversed the antinociceptive effects of baicalin. Compared with the saline group, the expression of α2a-AR and α2c-AR mRNA was upregulated significantly in the baicalin group (P<0.05). Levels of α2-AR mRNA were also decreased in the baicalin + idazoxan group compared with the baicalin group (P<0.05). The levels of TNF-α, IL-6, IL-17 and IL-1β were raised after treatment with baicalin. In addition, baicalin treatment ameliorated the histological damage in the spinal cord. The percentage of CD4+ PBMCs was increased in the saline group compared with the control group (P<0.05). Compared with the baicalin group, the percentage of CD4+ PBMCs was raised after treatment with the α2-AR antagonists. In conclusion, intrathecal injection of baicalin produced an antiallodynic effect in a spinal nerve ligation-induced neuropathic pain model. The mechanism may be related to the regulation of a2-AR expression.


Introduction
Neuropathic pain is one of the most common categories of chronic pain and sensory dysfunction affecting a considerable proportion of the global population, negatively influencing their emotional health and overall quality of life (1). The prevalence of trauma related to the peripheral nervous system in the United States is 1.3-2.8% (2). Neuropathic pain is characterized by a response to non-noxious stimuli (tactile allodynia), spontaneous pain (exaggerated pain response to normal painful stimulus), hyperalgesia and loss of sensation in local areas (3,4). It can be triggered or initiated in the peripheral or central nervous system (5). Owing to the complex etiology of neuropathic pain, it is considered to be one of the most challenging pathologies to treat in clinical practice (6). At present, most pain medications to treat neuropathic pain are not satisfactory, and cause undesirable side effects. Therefore, it is necessary to find an effective therapy with minimal side effects.
It is well known that α 2 -adrenoceptor (α 2 -AR) agonists have antinociceptive effects in the spinal cord (7). α 2 -ARs are located not only in the central nervous system, but also in the peripheral nervous system (8). The complex processes involved in α 2 -AR pain regulation have been extensively researched. A previous study demonstrated that α 2 -AR agonists could relieve nerve injury-induced pain by binding to α 2 -AR in patients and animal models with neuropathic pain (9). Blockade of spinal 5-hydroxytryptamine (HT)3 receptors reduced α 2 -AR-mediated anti-hypersensitivity by reducing total GABA release (10). Furthermore, it is well known that α 2 -AR agonists can enhance the analgesic effects of morphine (11). Recently, the α 2 -AR antagonists atipamezole and idazoxan have been shown to block the induction of tolerance to morphine, which was verified through intrathecal atipamezole in opioid naïve and tolerant rats weakening the anti-nociceptive effect of morphine (12).

Baicalin relieves neuropathic pain by regulating α 2 -adrenoceptor levels in rats following spinal nerve injury
Baicalin is a common flavonoid substance isolated from the root of Scutellaria baicalensis Georgi. Previous studies have demonstrated that baicalin possesses anti-inflammatory, antioxidant, antitumor and antiallergy properties (13)(14)(15). Furthermore, Chou et al (16) established the model of carrageenan-evoked thermal hyperalgesia in rats and found that baicalin had a clear analgesic effect. A previous study also demonstrated that baicalin helps relieve pain in patients suffering from osteoarthritis of the knee (17).
In this study, it was determined whether baicalin may reduce pain in a spinal nerve ligation rat model of neuropathic pain, and the roles of the peripheral α 2 -AR subtypes in the mechanism of action of baicalin were investigated. Neuropathic pain model. Animals were anesthetized with halothane, 1-3% in oxygen, with spontaneous ventilation. A 3 cm paramedian incision was made in the left L4-sacral area, and a bundle of paraspinal muscles was removed to visualize the left L6 transverse process. Using small scissors, the left L6 transverse process was removed completely and the L4-L5 spinal nerves were exposed. After the L4 spinal nerve was separated, the L5 spinal nerve was cut and spread laterally. The fascia and skin were closed using sutures, and the animals were allowed to recover for 10 days prior to the epidural catheterization. Paw withdrawal mechanical threshold (PWT) <4 g after surgery was recognized as the standard of neuropathic pain induction.

Animals
Drugs administration. A total of 70 rats were randomly divided into the following groups: Control group (n=10); saline group (n=10); baicalin group (n=10); baicalin combined α 2 -AR antagonist groups (n=40). The α 2 -AR antagonist group was subcategorized in to four groups based on the antagonist used, which included the nonselective α 2 -AR antagonist idazoxan (n=10); α 2a -AR antagonist BRL 44408 (n=10); α 2b -AR antagonist ARC 239 (n=10); and α 2c -AR antagonist JP 1302 (n=10). The rats were treated with 20 mg/kg baicalin by intrathecal injection. The drugs used in the study were purchased from Tocris Bioscience. Idazoxan was dissolved in distilled water, and the other drugs were dissolved in physiological saline. All drugs were delivered in a volume of 2 µg/20 µl administered by intrathecal injection. In the control group, the rats did not undergo any surgery. The rats in saline group were injected with physiological saline (10 µl). Drugs were administered once per day for 7 days.
Behavioral tests. The PWT was measured by the up and down method (18). A series of von Frey filaments (0.4, 0.7, 1.2, 2.0, 3.6, 5.5, 8.5 and 15 g) in a perpendicular fashion were used to stimulate the surface of the lateral paw. Each was applied until slightly bent and held for approximately 5 sec. Reponses in the form of sharp withdrawal or paw licking were regarded as a positive response. Only rats with marked allodynia (withdrawal threshold <4 g) after spinal nerve ligation were studied.
Expression of α 2 a-AR, α 2 b-AR, α 2 c-AR mRNA in the spinal cord. When the final test was completed, three rats from each experimental group were sacrificed by cervical dislocation whilst anesthetized to obtain the L4-L5 dorsal spinal cord. Tissue samples were frozen immediately at -80˚C. Total RNA in spinal cord tissues was extracted using an RNeasy kit (cat. no. 74104; Qiagen GmbH) following the manufacturer's instructions. RNA quality and quantity were measured using a Nanodrop Spectrophotometer (UL-2000; Macylab Instruments, Inc.), while RNA integrity was assessed by gel electrophoresis. A total of 500 ng RNA was used to generate cDNA with a reverse transcription (RT) kit (Takara Biotechnology Co., Ltd.). The temperature conditions for the RT procedure were 65˚C for 10 min, 37˚C for 10 min, 75˚C for 15 min and 37˚C for 20 min. A Mastercycler ® nexus X2 (Eppendorf) was used for RT-quantitative (q) PCR (Power SYBR™ Green RNA-to-C T ™ 1-step kit, cat. no. 4389986; Thermo Fisher Scientific, Inc.). The thermocycling conditions were 95˚C for 15 sec followed by 35 cycles of 95˚C for 15 sec and 60˚C for 1 min. The relative levels of target mRNAs were standardized to the reference gene β-actin gene. The results were quantified using the 2 -ΔΔCq method (19). Primers for RT-qPCR in this study were as follows: Histological evaluation. Spinal cord tissues were fixed with 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd.) at 37˚C for 24 h, dehydrated and then embedded in paraffin wax. The tissues were then cut into 5-µm-thick sections, and stained with hematoxylin and eosin solution (Fuzhou Maixin Biotech Co., Ltd.) at 37˚C for 5 min. Pathological changes were observed under a light microscope (magnification, x400; Nikon Corporation).
Flow cytometry analyses. The peripheral blood mononuclear cells (PBMC) were separated from blood following the Ficoll 400 and uropolinum density centrifugation method (20). The T-lymphocyte subset CD4 + was identified by flow cytometry analysis of cells isolated from the PBMCs. PBMCs adjusted to a density of 1x10 6 cells/ml in complete medium were used for analysis. Phytohemagglutinin solution (25 µl; Cylex, Inc.) was added to block non-specific binding and the cells were incubated for 15-18 h at 37˚C and 5% CO 2 .
The frequency of the T-lymphocyte subset was evaluated after staining with the FITC-conjugated mouse anti-rat CD4 (1:100; cat. no. FAB554F, BD Biosciences) at 4˚C for 25 min. After washing, cells were incubated with the pacific blue-A fluorochrome-conjugated isotype control (1:100; cat. no. A10478,  Statistical analysis. Statistical analysis was implemented using SPSS 20.0 (IBM Corp.). Statistical comparisons between groups were analyzed using one-way ANOVAs followed by Duncan multiple range post hoc tests. All results are reported as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference. Each test was repeated 3 times.

Results
Baicalin increases the PWT. Compared with the control group, a significant decrease in PWT was observed in all treatment groups (P<0.05; Fig. 1). After baicalin treatment, PWT increased, but the antiallodynic effect of baicalin was antagonized by intrathecal injection of α 2 -AR antagonists. PWT was reduced after 5 days of idazoxan administration when compared to baicalin group (P<0.05).
Histopathological changes in spinal cord tissue. As shown in Fig. 4A, the distribution of spinal cord neurons was orderly, and the nuclei were clearly visible in the control group. However, the number of neurons decreased and the distribution of neurons was uneven in the saline group (Fig. 4B). Compared with the saline group, baicalin treatment decreased neuronal apoptosis and reversed the pathomorphology (Fig. 4C). Intrathecal administration of different α 2 -AR antagonists reversed the effects of the baicalin treatment (Fig. 4D-G).

Baicalin decreases the expression of CD4 + cells.
To analyze the effects of α 2 -AR expression on CD4 + T cells, the percentage of CD4 + PBMCs was measured by flow cytometric analysis (Fig. 5). The results showed that the frequency of CD4 + cells was significantly increased in rats following spinal nerve injury (P<0.05). Compared with the saline group, baicalin treatment suppressed the frequency of CD4 + cells (P<0.05). The administration of different α 2 -AR antagonists appeared to increase the number of CD4 + cells compared with that in baicalin group but this difference was not significant.

Discussion
Previous studies have shown that peripheral administration of an α 2 -AR agonist attenuates nociceptive responses in both control animals and hypersensitive animals under neuropathic conditions (20,21). The results of this present study showed that intrathecal injection of baicalin attenuated neuropathic pain induced by spinal cord ligation, and the antiallodynic effects of baicalin were attenuated by α 2 -AR antagonists. This present study revealed that baicalin relieved pain by reducing inflammation, and this beneficial effect may be associated with the expression of α 2 -AR in spinal cord.
A previous study reported that norepinephrine and other α 2a -AR agonists decreased the release of glutamate in healthy rat dorsal horn synaptosomes, and had analgesic as well as anti-sympathetic effects (22). α 2c -AR was recognized to contribute to spinal cord analgesia induced by α 2 adrenoreceptor agonists (23). Blockade of spinal 5-HT3 receptors reduced α 2 -adrenoceptor-mediated anti-hypersensitivity via reducing total GABA release (24). α 2 -AR stimulation induces Gs-mediated acetylcholine release in the dorsal horn after peripheral nerve injury (25). In this present study, the expression levels of α 2 -ARs were changed in spinal nerve injury rats, compared to untreated rats. Notably, intrathecal administration baicalin increased α 2a and α 2c -AR mRNA. These results indicated that baicalin relieved the pain by regulating α 2 -AR mRNA levels. There is increasing evidence demonstrating that neuroinflammation is one of the pivotal contributors to the development of neuropathic pain. Some pro-inflammatory cytokines produced by microglia in the spinal cord, such as IL-6, IL-17 and IL-1β, play an important role in inflammatory processes (26). TNF-α is also a biomarker of acute neuro-inflammatory responses (27). The present study showed that the serum levels of TNF-α, IL-6, IL-17 and IL-1β increased after spinal nerve ligation; however, the release of TNF-α, IL-6 IL-17 and IL-1β was reduced by intrathecal administration of baicalin. Furthermore, baicalin treatment appeared to improve the order of nerve fibers and reduced the percentage of CD4 + PBMCs. These data suggested that the baicalin was capable of reducing neuropathic pain by regulating the inflammatory response.
In conclusion, this present study indicated that intrathecal administration of baicalin relieved neuropathic pain following spinal nerve ligation in rats. The mechanisms of action may be through upregulating the expression of α 2 -ARs in the spinal cord. This may suggest that baicalin has therapeutic potential for neuropathic pain.

Funding
This work was supported by Nature Science Foundation of Shandong Province (grant no. ZR2015HL029) and Science and Technology Planning Project of Yantai Urban (grant nos. 2014WS046 and 2015WS070).

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions
LJH and SSJ participated in the design of the study and XHS, XYL, FFW, WL and QSJ carried out the study and performed statistical analysis. LJH, and SSJ drafted the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate
All experiments in this study were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The procedures were all approved by animal Ethics Committee of China Pharmaceutical University.

Patient consent for publication
Not applicable.