Introduction

Neuropathic pain is caused by injury or disease of the somatosensory nervous system, which ultimately causes an increased response to painful stimuli (hyperalgesia), unpleasant and abnormal sensation (dysesthesia), and pain in response to a stimulus that does not normally provoke pain (allodynia) (Finnerup et al., 2016; Kumar et al., 2017).Neuropathic pain is a major public health problem as it has a considerable impact on quality of life for patients (Elzahaf et al., 2016; Burke et al., 2017). Therefore, development of novel effective analgesics for neuropathic pain relief and treatment is warranted.

Transient receptor potential (TRP) ion channels constitute a major class of calcium-permeable, non-selective cation channels (Montell and Rubin, 1989; Gonzalez-Ramirez et al., 2017). TRP channels are characterized by various activation mechanisms (Nilius, 2007; Balemans et al., 2017). Some of the TRP ion channel superfamily are reportedly involved in nociception and thermosensation, in both the peripheral and central nervous system (Caterina et al., 1997; Moore et al., 2018). TRP vanilloid 1 (Trpv1) is activated by multiple mechanisms, such as noxious heat (＞ 43°C), protons, and pungent chemicals (e.g., capsaicin) (Caterina et al., 1997; Caterina and Julius, 2001). TRP ankyrin 1 (Trpa1) is expressed in sensory neurons, which are involved in the pain pathway(Naziroglu and Braidy, 2017). Trpa1 functions as a sensor of environmental and endogenous chemical irritants such as acrolein, 4-hydroxynonena, and allyl isothiocyanate (Cho et al., 2012). Trpv1 and Trpa1 play a crucial role in nociceptive transmission under pathological forms of pain (Lappin et al., 2006; Spicarova and Palecek, 2008; Chen et al., 2009).Moreover, spinal synaptic plasticity accounts for the transi-tion from acute to chronic pain, in which Trpv1 and Trpa1 play critical roles presynaptically and postsynaptically (Choi et al., 2016). Trpv1 and Trpa1 expression in nociceptors is altered in different models of neuropathy under pathological conditions (Biggs et al., 2007; Kim et al., 2008).

Natural plant products are traditional Chinese medicines for the therapy of chronic comorbidities (e.g., diabetes, cancer, and pain) (Yuan et al., 2015; Hager et al., 2016; Tsai et al., 2017). Among the compounds examined, puerarin was isolated from Radix Puerariae (Zhou et al., 2014). Since then, the pharmacological properties of puerarin have been widely investigated. Puerarin is commonly used in the treatment of myocardial and cerebral ischemia (Tao et al., 2017;Xiao et al., 2018). Puerarin also serves as a potent antioxidant and anti-in fl ammatory agent (Kim et al., 2010; Zhao et al., 2016). Moreover, puerarin alleviates diabetic neuropathic pain by regulating P2X3 receptor expression in dorsal root ganglion (DRG) neurons (Xu et al., 2012). Puerarin plays a neuroprotective effect on acute spinal cord injury (Zhang et al., 2016). Altogether, these studies suggest a potential role of puerarin in relief of pain originating from the central or peripheral nervous system.

The putative impact of puerarin on neuropathic pain and the underlying mechanisms remain unclear. Consequently,we hypothesized that puerarin may ameliorate neuropathic pain via Trpv1 or Trpa1. Here, we investigated the analgesic role and possible mechanisms of puerarin on neuropathic pain using animal behavioral tests and molecular biological methods. Thus, our aim in the present study was to examine the effect of puerarin on neuropathic pain.

无论裂隙位于哪个位置，边坡安全系数总是随着裂隙深度的增加而较小的。膨胀土边坡在经历干湿循环的过程中不断发生胀缩变形，而由于土体表面和内部含水率不一致导致其胀缩程度不一致，土体胀缩不均，于是产生裂隙。当裂隙底部成为新的蒸发面以后，以上过程又会重复发生，导致裂隙不断扩展、深入。降雨初期，雨水因土体出现的短暂疏水性而在地表累积，一旦形成积水，雨水会优先沿着裂隙侧壁入渗[21]，汇聚在裂隙底部，之后入渗至土体内部。因此，裂隙的深度直接控制了膨胀土边坡内部的饱和区范围。深度越大，降雨后沿着裂隙进入膨胀土边坡的雨水越多，形成的饱和区域越大，对膨胀土边坡的稳定性越不利。

Materials and Methods

Animals

Twenty-eight male Sprague-Dawley rats weighing 200—220 g were purchased from the Experimental Animal Center of Wuhan University of China (SYXK (E) 2014-0013). Rats were housed with 2—3 rats/cage under a standard 12-hour light/dark cycle at 23 ± 1°C, and allowed free access to water and chow.All rats were acclimated to the experimental circumstances for a week before experiments. All experimental procedures were performed in accordance with the guidelines of the International Association for the Study of Pain, and approved by the Institutional Animal Care and Use Committee of Renmin Hospital of Wuhan University of China (WDRMSPF/SQ-26).

Financial support: This study was supported by the National Natural Science Foundation of China, No. 81671891. The funding body played no role in the study design, in the collection, analysis and interpretation of data, in the writing of the paper, and in the decision to submit the paper for publication.

In conclusion, cumulative puerarin (30 and 60 mg/kg)administration (intraperitoneally) mitigated allodynia and hyperalgesia induced by pSNL. Thus, in the current study,we provide novel evidence suggesting that Trpv1 and Trpa1 in DRG contribute to the analgesic role of puerarin in neuropathic pain. Taken together, puerarin plays an important analgesic role in neuropathic pain and might serve as a potential compound for clinical treatment of neuropathic pain.

Surgery

All surgeries were performed with the rats under deep anesthesia induced by intraperitoneal injection of sodium pentobarbital (50 mg/kg). To create the neuropathic pain model, partial sciatic nerve ligation (pSNL) was performed as described previously (Shir and Seltzer, 1991). In brief, a tight ligation of approximately one-third to one-half the diameter of the right sciatic nerve (ipsilateral) was performed using a 6-0 silk suture, as described previously (Seltzer et al., 1990). In sham-operated rats, the nerve was exposed without ligation. Behavioral tests were performed to con fi rm the success of pSNL.

Drug administration and experimental design

All behavioral tests were performed during the day, before and after pSNL every day. Rats were habituated to the testing apparatus for at least 30 minutes before behavioral testing. Von Frey fi laments were applied with increasing force(from 0.4 to 60.0 g) until the paw withdrawal threshold was detected (Wang et al., 2011). Briefly, von Frey hairs were pressed vertically onto the hind plantar surface for approximately 4—5 seconds. Each fi lament was used ten times, with a 5-minute interval between different forces. The minimal force that caused a lifting or licking response at least fivetimes was considered the paw withdrawal threshold.

Nociceptive tests Mechanical allodynia

Puerarin was dissolved in 10% dimethyl sulfoxide diluted with 0.9% saline. Stock solution was fi ltered through a 0.22 μm membrane before use. Rats were randomly divided into four groups (n = 7): sham group, pSNL group, pSNL +puerarin 1 group (30 mg/kg, intraperitoneally), and pSNL+ puerarin 2 group (60 mg/kg, intraperitoneally). Rats received intraperitoneal injection of puerarin daily for 7 consecutive days, while sham and pSNL rats received the same volume of dimethyl sulfoxide in saline.

Thermal hyperalgesia

Heat sensitivity was examined using a Hargreaves radiant heat apparatus (Ugo Basile, Milan, Italy). Rats were acclimated in a plastic cage for 30 minutes before testing. The cage was then placed on a glass plate above the plantar test apparatus and a movable noxious heat source placed directly under the plantar surface of the hind paw (Cherng et al.,2014). When activated, the apparatus applied a continuous infrared heat stimulus to the plantar surface, which elicits a distinctive paw withdrawal reflex and stops an automated timer (based on infrared re fl ection). Each rat was measured three-times separated by a 10-minute interval. The average value was used as the response latency.

Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR)

Rats were sacrificed after pSNL. Naive rats were used as controls. Lumbar 4, 5, and 6 DRGs were collected both ipsilaterally and contralaterally, with respect to the injury side. DRGs from all three levels were pooled from each side and placed into tubes with Trizol reagent (Sigma-Aldrich,St. Louis, MO, USA). RNA isolation was followed by chloroform extraction and isopropanol precipitation. Extracted RNA was dissolved in 20 μL RNase-free water and stored at −80°C. cDNA was synthesized using the iScript™ cDNA Synthesis kit (Biorad, Hercules, CA, USA). Copy number of rat genes was determined by RT-PCR using SYBR Green MASTER (Invitrogen, Waltham, MA, USA), following the manufacturer’s protocol. Data were collected during each extension phase of PCR and analyzed using ABI-7700 SDS software (Applied Biosystems, Foster City, CA, USA). RNA levels were normalized to β-actin levels, and calculated by delta-delta threshold cycle (ΔΔCT). Primers were used according to previous studies (Xue et al., 2007; Pan et al., 2014;Pohoczky et al., 2016). Primer sequences are shown in Table 1.

Statistical analysis

Data are presented as mean ± SEM. Statistical analysis was performed using repeated measures analysis of variance followed by the least significant difference post hoc test or one-way analysis of variance followed by Bonferroni correction using Origin 10.0 software (OriginLab Corporation,Northampton, MA, USA). Only two-tailed P values less than 0.05 were considered statistically signi fi cant.

心内科专科护士培训的最终目标是提高受训护士的临床护理能力，使其尽早成为合格的心内科护士。构建专科护士培训SOP的目的是促进培训的系统、规范及高效化，同时加强对专科培训的监督和管理，提高专科培训质量。中国医科大学附属第一医院心内科护理团队采用SOP方法在心内科专科护士培训中进行了初步探索并取得了良好效果，但如何持续改进SOP方法并不断提高专科护士培训质量仍是今后需要探索的问题[14-15]。

Con fl icts of interest: None declared.

Results

Establishment of a rat model of neuropathic pain

After surgery, mechanical threshold and paw withdrawal latency was tested in rats from sham and pSNL groups. pSNL model rats showed mechanical sensitivity at 1 day after pSNL surgery(P ＜ 0.01; Figure 1A). Simultaneously, signi fi cant thermal hyperalgesia to noxious heat stimulation was observed in pSNL model rats (P ＜ 0.01; Figure 1B). These results suggest that the pSNL model has been successfully established in rats.

Puerarin ameliorates mechanical allodynia in neuropathic pain rats

Next, we investigated the analgesic effect of puerarin by intraperitoneal administration in rats with peripheral nerve injury.Our results show that the mechanical threshold (ipsilateral)was signi fi cantly increased in both the pSNL + puerarin 1 and pSNL + puerarin 2 groups compared with the pSNL group (P＜ 0.05 or P ＜ 0.01; Figure 2A). Moreover, in rats at 7 days after pSNL, maximal efficiency and ratio of response size of mechanical allodynia after 7-day puerarin treatment compared with no puerarin treatment showed that puerarin dose-dependently relieved neuropathic pain (P ＜ 0.01; Figure 2B). These results show that puerarin dose-dependently ameliorates mechanical allodynia in rats after peripheral nerve injury.

Puerarin mitigates thermal hyperalgesia in neuropathic pain rats

Paw withdrawal latency upon thermal stimulation was examined in rats. Puerarin also reversed thermal hyperalgesia after only one-day of injection in pSNL model rats (P＜ 0.05 or P ＜ 0.01; Figure 3A). Moreover, in rats at 7 days after pSNL, maximal efficiency and ratio of response size of thermal hyperalgesia after 7-day puerarin treatment compared with no puerarin treatment also showed that puerarin dose-dependently relieved neuropathic pain (P ＜ 0.05 or P ＜0.01; Figure 3B). This indicates that puerarin reduces thermal pain. Altogether, our data suggest that puerarin plays a notable analgesic role in neuropathic pain.

Puerarin suppresses increased Trpv1 and Trpa1 mRNA expression in DRG of neuropathic pain rats

Quantitative RT-PCR was performed to investigate the potential mechanism involving puerarin. We examined wellknown cation channels that are expressed in nociceptiveneurons. Both Trpv1 and Trpa1 mRNA expression levels were significantly increased in DRG of neuropathic pain model rats after injury (P ＜ 0.01; Figure 4A and B). In contrast, throughout 7 days of puerarin administration, Trpv1 and Trpa1 mRNA expression was significantly reversed in DRG of pSNL model rats (P ＜ 0.05 or P ＜ 0.01; Figure 4A and B). These results show that puerarin relieves neuropathic pain by inhibiting Trpv1 and Trpa1 up-regulation in DRG of neuropathic pain rats.

Table 1 Sequences of primers for quantitative real-time reverse transcriptase-polymerase chain reaction

Trpv1: Transient receptor potential vanilloid 1; Trpa1: transient receptor potential ankyrin 1.

Product size (bp)GeneSequence (5′–3′)Trpv1Forward: AAT ACA CCA TCG CTC TGC T19 Reverse: CAA TGT GCA GTG CTG TCT GG20 Trpa1Forward: AGT GGC AAT GTG GAG CGA TA20 Reverse: TCC CGT CGA TCT CAG CAA TG20 β-Actin Forward: AAG TCC CTC ACC CTC CCA AAA G 22 Reverse: AAG CAA TGC TGT CAC CTT CCC21

Discussion

Neuropathic pain is characterized by hyperalgesia, allodynia,altered sensation, and spontaneous pain. Once a peripheral nerve is damaged, a series of pathophysiological events are induced, including changes in perineuronal homeostasis, neuronal hyperexcitability, alterations in gene expression and the immune response (Hanani, 2012; Ji et al., 2016). Meanwhile,subsequent events in central pain processing pathways consolidate and exaggerate the steady-state pain condition (Kuner, 2010; Kumar et al., 2017). Neuropathic pain is challenging to manage, and current treatment strategies often lack efficacy or have severe side-effects in most patients (Artemiadis and Zis, 2018). In the present study, we show that puerarin isolated from Radix Puerariae ameliorates neuropathic pain. Puerarin injection also reversed Trpv1 and Trpa1 mRNA up-regulation in the DRG of rats with peripheral nerve injury. These results suggest that puerarin may play a promising analgesic role by preventing up-regulation of Trpv1 and Trpa1 in the DRG of neuropathic pain animals.

Institutional review board statement: This study was approved by the Institutional Animal Care and Use Committee of Renmin Hospital of Wuhan University of China (approval No. WDRMSPF/SQ-26). The experimental procedure followed the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1985).

某校高一年级共有20个班，每班有50名学生．为了了解高一学生的视力状况，从这1 000人中抽取一个容量为100的样本进行检查，该怎样检查？

Trpv1 and Trpa1 are well-known channels involved in nociception and thermosensation, both in the peripheral and central nervous system (Caterina et al., 1997). Trpv1 and Trpa1 play a crucial role in nociceptive transmission under pathological forms of pain (Lappin et al., 2006; Spicarova and Palecek, 2008; Chen et al., 2009). Moreover, it is clear that spinal synaptic plasticity accounts for the transition from acute to chronic pain, in which Trpv1 and Trpa1 play critical roles presynaptically and postsynaptically (Yuan and Burrell, 2010; Jensen and Edwards, 2012; Choi et al., 2016).Trpv1 and Trpa1 expression in nociceptors is altered under pathological conditions in different models of neuropathy(Biggs et al., 2007; Kim et al., 2008). Here, our results show that consecutive puerarin administration ameliorates me-chanical allodynia and thermal hyperalgesia in rats with peripheral nerve injury. Further, cumulative puerarin markedly recovered mechanical threshold and paw withdrawal latency (in response to thermal stimulation) in a dose-dependent manner. Altogether, these results suggest that puerarin may be an effective anti-nociceptive agent. To further con fi rm the analgesic role of puerarin in neuropathic pain,it is necessary to examine its effect in different neuropathic pain models.

Figure 1 Mechanical and thermal tests in rats with peripheral nerve injury.

Mechanical threshold (A) and paw withdrawal latency (B) in response to thermal stimulation in rats after pSNL. **P ＜ 0.01, vs. sham group at the same time point (mean ± SEM, n = 7; repeated measures analysis of variance followed by least signi fi cant difference post hoc test). pSNL: Partial sciatic nerve ligation.

Figure 2 Analgesic role of puerarin on mechanical allodynia in rats with peripheral nerve injury.

(A) Mechanical allodynia in rats treated with or without puerarin (intraperitoneally, 30 or 60 mg/kg) after pSNL. (B) Maximal efficiency and ratio of response size of mechanical allodynia in rats at 7 days after pSNL and after 7-day puerarin treatment compared with no puerarin treatment.†P ＜ 0.05, ††P ＜ 0.01, vs. pSNL group; ##P ＜ 0.01, vs. pSNL + puerarin 1 group at the same time point (mean ± SEM, n = 7). Statistical analysis was performed with repeated measures analysis of variance followed by least signi fi cant difference post hoc test (A) or one-way analysis of variance followed by Bonferroni correction (B). pSNL + puerarin 1 group and pSNL + puerarin 2 group: each rat was administrated 30 and 60 mg/kg (intraperitoneally) for 7 days after surgery. pSNL: Partial sciatic nerve ligation.

Figure 3 Analgesic effect of puerarin on thermal hyperalgesia in rats with peripheral nerve injury.

(A) Thermal hyperalgesia in rats treated with or without puerarin (intraperitoneally, 30 or 60 mg/kg) after pSNL. (B) Maximal efficiency (%) and ratio of response size of thermal hyperalgesia in rats at 7 days after pSNL and after 7-days puerarin treatment compared with no puerarin treatment. †P ＜ 0.05, ††P ＜ 0.01, vs. pSNL group (mean ± SEM, n = 7; repeated measures analysis of variance followed by least signi fi cant difference post hoc test); ##P ＜ 0.01, vs. pSNL + puerarin 1 group at the same time point (mean ± SEM, n = 7). Statistical analysis was performed with repeated measures analysis of variance followed by least signi fi cant difference post hoc test (A) or one-way analysis of variance followed by Bonferroni correction (B). pSNL: Partial sciatic nerve ligation.

Figure 4 mRNA expression levels of Trpv1 and Trpa1 in dorsal root ganglion of pSNL model rats after puerarin treatment.

Trpv1 (A) and Trpa1 (B) mRNA expression levels relative to levels in the sham group in the dorsal root ganglion of pSNL model rats after puerarin treatment for 7 days. **P ＜ 0.01, vs. sham group. †P ＜ 0.01; ††P ＜ 0.01, vs. pSNL group (mean ± SEM, n = 7; one-way analysis of variance followed by Bonferroni correction). pSNL: Partial sciatic nerve ligation; Trpv1: transient receptor potential vanilloid 1; Trpa1: transient receptor potential ankyrin 1.

Mechanisms of the analgesic role of puerarin in neuropathic pain are still not clear. Puerarin can alleviate diabetic neuropathic pain by regulating expression of purinoceptor 3(P2X3) in DRG neurons (Li et al., 2011; Xu et al., 2012; Liu et al., 2014b). Moreover, puerarin alleviates neuropathic pain by inhibiting neuroinflammation in the spinal cord (Liu et al., 2014a). Puerarin also exerts a neuroprotective effect in acute spinal cord injury rats by decreasing neuronal loss, inhibiting glial cell activation, and alleviating in fl ammation in the injured spinal cord. In addition, downregulated phosphoinositide 3-kinase and phospho-Akt protein expression was restored by puerarin in acute spinal cord injury rats (Zhang et al., 2016). Our present observations demonstrate that the natural plant product, puerarin, largely prevents up-regulation of Trpv1 and Trpa1 mRNA in DRG neurons of neuropathic pain rats in a dose-dependent manner. These results suggest that the analgesic role of puerarin may act by suppressing Trpv1 and Trpa1 up-regulation. Furthermore, puerarin modulates expression of protein kinase C (Zhu et al., 2008; Tang et al., 2014), which acts upstream of Trpv1 and Trpa1 (Tominaga, 2010; Mandadi et al., 2011; Ozdemir et al., 2016; Simoes et al., 2016). Therefore, it is possible that puerarin prevents Trpv1 and Trpa1 up-regulation through protein kinase C modulation. Further studies are warranted in the future.

主流意识形态认同是社会成员对主流意识形态的承认、接受、和共享，具体体现在主体对主流意识形态的认知、情感和评价等话语体现。目前，我国社会的主流意识形态主要存在政治话语、学术话语和大众话语三种形式，这三种话语在网络空间中形成相互交织的共存状态。可“微时代”挤压了大学生主流意识形态认同的空间，所以大学生对政治缺乏热情和兴趣。同时，学术话语太抽象，除了特别专业的学生学习之外，大部分学生都不感兴趣，几乎没有人会对它提出自己的看法。此外，“微文化”碎片化、大众化、快餐化的特点导致了微文化的虚假性，它的娱乐性吸引了大学生，缩小了大学生对主流意识形态空间的认同。

Author contributions: ZYX designed the study. HTX, XP, BZ and ZGL performed experiments. HTX analyzed data. HTX and ZYX wrote the paper. All authors approved the fi nal version of the paper.

很快选好一款跑步鞋，售货员小伙子说，如果扫码加品牌微信号，鞋子即可五折，如果不扫，则是七折。扫个码便宜两成，我想这划得来。扫完码之后，手机屏幕出现注册画面，要求填写姓名、性别、年龄、手机号；因为扫了码，手机号码已经自动出现在注册栏里。这一下引起了我的警觉。姓名、年龄、性别、手机号已是我的基本身份信息，依据这些信息甚至可以在网上对我进行定位跟踪。

转基因食品的销售层面：在销售过程中，销售者应记录每一批产品的来源地及去向地，以及每一地区的负责人等信息，并妥善保管，以备查阅。

适宜的音乐刺激听觉中枢，使人体交感神经系统活动减弱，副交感神经活动增强，从而能让患者在应激下保持平稳状态［17］。音乐疗法可显著降低患者的激素水平［18-19］、心率、呼吸频率和舒张压。研究发现，音乐干预对心率影响较小［20-21］，可降低舒张压约 2 mm Hg，但对收缩压没有作用［13］。Ni等［22］观察发现音乐干预对生命体征的影响非常轻微，还有报道指出音乐干预对收缩压和心率没有影响，但音乐有益于安抚待手术患者的情绪，能改善患者的手术体验和提高疗效。

Copyright license agreement: The Copyright License Agreement has been signed by all authors before publication.

Data sharing statement: Datasets analyzed during the current study are available from the corresponding author on reasonable request.

Plagiarism check: Checked twice by iThenticate.

Peer review: Externally peer reviewed.

Open access statement: This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

Open peer reviewer: Tufan Mert, Department of Biophysics, School of Medicine, University of Cukurova, Turkey.

Additional fi le: Open peer review report 1.

学习模式分为大课与小课，大课在人口集中的辖区内举行，地方合作组织单位领导对参训学员进行政策性就业指导与培训就业动员，要求学员严格按照课程设置配合学校教学工作，珍惜学习机会，练就专业技能本领，争取早日走上工作岗位，促进家庭经济增收。

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