Effect of Peptidase Inhibitors on Dynorphin A (1-17) or (1-13)-Induced Antinociception and Toxicity at Spinal Level

Our group has earlier demonstrated that three enzymes sensitive to peptidase inhibitors (PIs), amastatin (A)-, captopril (C)-, and phosphoramidon (P), played an important role in inactivation of enkephalins at the spinal level. Dynorphin-converting enzyme (DCE) hydrolyzes dynorphin (Dyn) A (1-17) or Dyn A (1-13) mainly at the Arg6-Arg7 bond. Dynorphin A and its derived peptides interact with opioid and glutamate receptors at their N- and C-terminals, respectively. The purpose of the present study was to evaluate the antinociceptive potency and toxicity of intrathecal administered Dyn A (1-17), Dyn A (1-13), or Dyn A (1-6) under pretreatment with ACP and/or the DCE inhibitor p-hydroxymercuribenzoate (PHMB). The effect of these PIs on Dyn A (1-17)-induced inhibition of electrically-evoked contractions in mouse vas deferens was also investigated. The inhibitory potency of Dyn A (1-17) on electrically-evoked contractions in mouse vas deferens under pretreatment with ACP was higher than that with AC, AP, or CP. Pretreatment with ACP augmented Dyn A (1-17) or (1-13)-induced antinociception by approximately 50- or 30-fold with no sign of allodynia when administered intrathecally at low doses. Pretreatment with ACP and PHMB induced neuropathy. These findings showed that intrathecal administration of low-dose Dyn A (1-17) or DynA (1-13) increased antinociception under pretreatment with ACP, but without signs of allodynia in rat.

Effects of moxibustion on dynorphin and endomorphin in rats with chronic visceral hyperalgesia

AIM:To observe the analgesic effects of moxibustion in rats with chronic visceral hyperalgesia and its influence on the concentration of dynorphin(Dyn) and endomorphin(EM) in spinal cord.METHODS:The rat model of chronic visceral hyperalgesia was established by colorectal distention(CRD).In moxibustion(MX) group,moxibustion was applied once daily for 7 d;in sham moxibustion(SM) group,moxibustion was given to the same acupoints but with the nonsmoldered end of the moxa stick.Model control(MC) group and normal control group were also studied.The scoring system of abdominal withdrawal reflex was used to evaluate visceral pain for behavioral assessment.Enzyme linked immunosorbent assay was performed to determine the concentrations of Dyn and EM in spinal cord.RESULTS:Moxibustion significantly decreased visceral pain to CRD in this rat model,and no significant difference was detected between the SM group and the MC group.In MX group,moxibustion also increased the concentrations of Dyn and EM in spinal cord,and no significant difference was found between the SM group and the MC group.CONCLUSION:Moxibustion therapy can significantly enhance the pain threshold of rats with chronic visceral hyperalgesia,and the effect may be closely related to the increased concentration of Dyn and EM in spinal cord.

A non-opioid pathway for dynorphin-caused spinal cord injury in rats

Intrathecal injection of dynorphin into rats via subarachnoid catheter induces damage to spinal cord tissue and motor function. Injection of the kappa opioid receptor antagonist nor-binaltorphine, or the excitatory amino acid N-methyl-D-aspartate receptor antagonist MK-801 into rats alleviated the pathological changes of dynorphin-caused spinal cord tissue injury and reduced the acid phosphatase activity in the spinal cord. The experimental findings indicate that there are opioid and non-opioid pathways for dynorphin-induced spinal cord injury, and that the non-opioid receptor pathway may be mediated by the excitatory amino acid N-methyl-D-aspartate receptor.

Immunohistochemical identification of dynorphin A and Kappa opioid receptor-1 in the digestive system of scallop Chlamys farreri

Little is known about the roles of dynorphin and Kappa opioid receptor(KOR) in mollusks. In this study, we aim to determine the distribution of dynorphin A and KOR-1 in the digestive system of the scallop Chlamys farreri. Using immunohistochemical staining, we confirmed the expression of dynorphin A and KOR-1 in the digestive system of C. farreri. Dynorphin A immunopositive cells were identified in intestine and hepatopancreas. In intestine, small and spherical dynorphin A immunopositive cells(4–9 μm in diameter) were scattered among the long columnar epithelial cells(ECs). In hepatopancreas, cells containing masses(5–14 μm in diameter) of dynorphin A immunopositive products were observed in epithelium of acinis. These immunopositive cells may be synthetic and/or secretory cells of dynorphin A. Dynorphin A immunoreactive products were commonly observed in epithelium and connective tissue(CT) of labial palps, mouth labia and stomach, which presented in forms of grains, fibers or flakes. KOR-1 immunoreactive material was observed in ECs and CTs of labial palps, mouth labia and stomach, intestine and hepatopancreas. The distribution of both dynorphin A and KOR-1 in the digestive organs suggests an involvement of dynorphin via KOR-1 in the functional regulation of the digestive system of C. farreri.

Kappa opioid receptor antagonist and N-methyl-D-aspartate receptor antagonist affect dynorphin-induced spinal cord electrophysiologic impairment

The latencies of motor- and somatosensory-evoked potentials were prolonged to different degrees, and wave amplitude was obviously decreased, after injection of dynorphin into the rat subarachnoid cavity. The wave amplitude and latencies of motor- and somatosensory-evoked potentials were significantly recovered at 7 and 14 days after combined injection of dynorphin and either the kappa opioid receptor antagonist nor-binaltorphimine or the N-methyl-D-aspartate receptor antagonist MK-801. The wave amplitude and latency were similar in rats after combined injection of dynorphin and nor-binaltorphimine or MK-801. These results suggest that intrathecal injection of dynorphin causes damage to spinal cord function. Prevention of N-methyl-D-aspartate receptor or kappa receptor activation lessened the injury to spinal cord function induced by dynorphin.

The aconitine analog bulleyaconitine A exhibits anti-hypersensitivity through direct stimulation of dynorphin A release from spinal microglia in the rat model of neuropathy

Aim Aconitine and its structurally-related diterpenoid alkaloids have been shown to interact differential- ly with neuronal voltage-dependent sodium channels and be responsible for their analgesia and toxicity. Bulleya- conitine A （ BAA or BLA） is an aconitine analog and has been prescribed for the management of pain. The present study aimed to evaluate the inhibitory effects of BAA on pain hypersensitivity and morphine anti-nociceptive toler- ance, and explore whether the release of dynorphin A from spinal microglia was associated with its mechanism of actions. Methods Rat models of neuropathic pain, formalin test and bone cancer pain were used, and spinal dynorphin A level and expression were measured. Sample size of animals was six in each study group. Resultes A single intrathecal or subcutaneous （but not intraventricular or local） injection of BAA blocked spinal nerve liga- tion-induced painful neuropathy, bone cancer-induced pain and formalin-induced hyperalgesia by 60% - 100% with the ED50 values of 94 - 126 ng/rat （intrathecal） and 42 - 59 μg · kg^-1 （ subcutaneous）, respectively. Follow- ing chronic treatment, BAA did not induce either self-tolerance to anti-nociception or cross-tolerance to morphine anti-nociception, and completely prevented morphine tolerance. Spinal BAA anti-nociception, but not neurotoxici- ty, was completely blocked by the specific microglial inhibitor minocycline. In a minocycline-sensitive and lido- BAA stimulated the release of dynorphin A from the spinal cord, and the caine- or ropivacaine-insensitive manner, primary culture of microglia but not from neurons or astrocytes. The blockade effects of BAA on nociception and morphine tolerance were completely blocked by the specific dynorphin A antiserum and/or K-opioid receptor antago- nist. Conclusions Our results demonstrated that BAA eliminated pain hypersensitivity and morphine tolerance through the direct stimulation of dynorphin A release from spinal microglia, which was not dependent on the interac- tions with sodium channels.

CHANGES OF PLASMA DYNORPHIN LEVELS BEFORE AND AFTER PERCUTANEOUS BALLOON MITRAL COMMISSUROTOMY IN PATIENTS WITH MITRAL STENOS

ＣＨＡＮＧＥＳＯＦＰＬＡＳＭＡＤＹＮＯＲＰＨＩＮＬＥＶＥＬＳＢＥＦＯＲＥＡＮＤＡＦＴＥＲＰＥＲＣＵＴＡＮＥＯＵＳＢＡＬＬＯＯＮＭＩＴＲＡＬＣＯＭＭＩＳＳＵＲＯＴＯＭＹＩＮＰＡＴＩＥＮＴＳＷＩＴＨＭＩＴＲＡＬＳＴＥＮＯＳＩＳＹｉｎＲｕｉｘｉｎｇ；（尹瑞...

RELATIONSHIP BETWEEN ACUPUNCTURE ANALGESIA AND MET-ENKEPHALIN OR DYNORPHIN

subjective: The effect of 4～5 Hz electroacupuncture (EA) on alterations of both met enkephalin (MEK) and dynorphin (Dyn) in the patient plasma or mouse spinal cord and its relation with analgesic effect were studied.Methods: In acupuncture clinic 10 patients with acute pain were treated with 4 Hz EA at Zusanli(ST 36) and/or Hegu(LI 4) acupoints for 30 min. 20 BALB/C mice were randomly divided into 2 groups: a. EA group(n=10), treated with 4～5 Hz EA at bilateral “Zusanli"(ST 36) for 15 min; b. control group(n=10) treated with no EA, but also restrained for 15 min. Before and after EA or restraining acupoints, the pain threshold of the patients or mice was detected. 10 μl of the patient plasma before and after EA and each mouse spinal cord suspension, of the 2 groups were blotted onto nitrocellulose membrane (NCM) respectively. The protein dot blot signals were detected by immunoreactivity (IR) and using Shimadu TLC Scanner and analyzed statistically. Results: The results showed that an increase in patient plasma MEK IR or Dyn IR and a decrease in mouse spinal MEK IR or Dyn IR could be detected, and the alteration of plasma or spinal MEK IR was more significant than that of plasma or spinal Dyn IR. There was a positive correlation in alteration between plasma or spinal MEK IR and plasma or spinal Dyn IR with respective parallel levels in individuals. The increased plasma MEK IR or the decreased spinal MEK IR was positively or negatively correlated with the analgesic effect, while the correlation between plasma or spinal Dyn IR and analgesic effect was insignificant. Conclusion: The results suggest that under lower frequency EA the met enkephalin may play an important role in analgesia.

EFFECTS OF DYNORPHIN A （1－17） ON MOTOR FUNCTION AND SPINAL INTRACELLULAR MESSENGER SYSTEMS IN RAT

ＥＦＦＥＣＴＳＯＦＤＹＮＯＲＰＨＩＮＡ（１－１７）ＯＮＭＯＴＯＲＦＵＮＣＴＩＯＮ ＡＮＤＳＰＩＮＡＬＩＮＴＲＡＣＥＬＬＵＬＡＲＭＥＳＳＥＮＧＥＲＳＹＳＴＥＭＳＩＮＲＡＴＺｈａｎｇＺｈｉｙｕａｎ；（张志媛），ＬｉＦｕｃｈｕｎ；（李富春），ＲｅｎＭｉｎｆ...

Levels of immunoreactive dynorphin A_(1-13) during development of morphine dependence in rats

目的：研究吗啡依赖大鼠组织内免疫活性强啡肽Ａ１－１３含量的动态变化及其与依赖程度的关系．方法：用纳洛酮催促的戒断症状评分测定吗啡依赖程度，用放射免疫测定组织内强啡肽Ａ１－１３水平．结果：在３－６天给药期内，吗啡可进行性降低脊髓、垂体、血浆内免疫活性强啡肽Ａ１－１３水平，升高海马及下丘脑的强啡肽Ａ１－１３水平，继续给药至１２天，各组织内免疫活性强啡肽Ａ１－１３水平不再有显著性变化．结论：吗啡依赖形成过程中脊髓、垂体及血浆内强啡肽Ａ１－１３呈进行性降低。

Involvement of dynorphin A in the inhibition of morphine physical dependence by N-nitro-L-arginine in rats

Objective To investigate the involvement of immunoreactive dynorphin A in the inhibitory effect of N nitro L arginine on the morphine physical dependence in rats Methods The rats were rendered dependent on morphine by subcutaneous administration of morphine solution three times daily in a manner of dose increment of 5 mg·kg 1 for 6 days The degree of morphine physical dependence was monitored by scoring the abstinence syndromes precipitated by 5 mg·kg 1 naloxone of the rats The expression levels of immunoreactive dynorphin A in tissues were determined using a radioimmunoassay Results Intraperitoneal injection of 5 mg·kg 1 N nitro L arginine suppresses most of the withdrawal symptoms of morphine dependent rats N nitro L arginine can elevate the expression of immunoreactive dynorphin Conclusions Chronic N nitro L arginine administration can inhibit the development of morphine physical dependence in a manner of dose dependence, which is significantly related to its role of regulating the endogeneous dynorphin system

生育舞蹈对初产妇产时疼痛、产程及分娩结局的影响

目的 探讨生育舞蹈对初产妇产时疼痛、产程及分娩结局的影响。方法 选择2022年1月至2023年3月在东莞市中西医结合医院分娩的140例初产妇为研究对象,根据随机数表法将所有产妇分为研究组和对照组,每组各70例。对照组产妇孕期行保健干预,研究组产妇在对照组的基础上行产时生育舞蹈干预。分娩后,比较两组产妇不同时间点的视觉模拟评分法(VAS)评分、总产程、产后2 h出血量、不同时间点强啡肽(DYN)水平及新生儿出生后1 min、5 min、10 min的Apgar评分。结果 镇痛前即刻,两组产妇的VAS评分比较差异无统计学意义(P>0.05);进入活跃期及宫口开全时,研究组产妇的VAS评分分别为(5.17±1.32)分、(6.43±0.64)分,明显低于对照组的(8.12±1.02)分、(8.54±0.83)分,差异均有统计学意义(P<0.05);研究组产妇的总产程、产后2 h出血量分别为(423.62±20.69) min、(213.61±25.82) m L,明显短(少)于对照组的(554.38±28.57) min、(314.76±28.71) m L,差异均有统计学意义(P<0.05);镇痛前即刻,两组产妇的DYN水平比较差异无统计学意义(P>0.05);宫口开全时,研究组产妇的DYN水平为(4.35±0.17) pg/mL,明显高于对照组的(3.08±0.18) pg/mL,差异有统计学意义(P<0.05);两组新生儿出生后1 min、5 min、10 min的Apgar评分比较差异均无统计学意义(P>0.05)。结论 生育舞蹈应用于初产妇可有效缓减产时疼痛,同时能提升DYN水平、缩短产程、减少产后出血量,具有临床推广价值。

Aladan scanning: The structure-activity relationship of dynorphin A

An unnatural amino acid, β-[6′-(N, N-dimethyl)amino-2′-naphthoyl]alanine (Ald) showing polarity-sen sitive fluorescence characteristics, was synthesized. A thorough Ald-scan of dynorphin A (Dyn A), the putative endogenous ligand for κ opioid receptors, was then performed. Replacement of the amino acid residues in positions 5, 8, 10, 12 or 14 of Dyn A(1-13)-NH2 with Ald resulted in compounds that had almost equal κ binding affinity compared with that of the parent compound; on the other hand, substi-tution of residues in position 1 or 4 with Ald decreased κ-receptor binding affinity. These results indi-cate that Tyr and Phe in Dyn A are very important for maintaining its κ-opioid activity. Evidence from receptor binding assay clearly displays that [Ald5]Dyn A(1-13)-NH2 is a highly selective κ-opioid re-ceptor agonist. An evaluation of the interaction of Ald-containing Dyn A(1-13)-NH2 analogues with SDS and DPC micelles was also performed. Interestingly, [Ald1]Dyn A(1-13)-NH2 and [Ald4]Dyn A(1-13)-NH2 showed quite different fluorescence emission maxima in SDS and DPC micelles. This indicates that both peptides are sensitive to electronic properties of the polar surface of the micelles.

Expression of preproopiomelanocortin mRNA and preprodynorphin mRNA in brain of spontaneously hyperte

目的：检测前阿黑皮原（ＰＯＭＣ）和强啡肽原（ＰＰＤ）基因在高血压（ＳＨＲ）和同龄ＷｉｓｔａｒＫｙｏｔｏ大鼠（ＷＫＹ）中的表达．方法：用地高辛（ＤＩＧ）标记的ＲＮＡ探针进行原位杂交检测ＰＯＭＣｍＲＮＡ和ＰＰＤｍＲＮＡ．结果：ＰＯＭＣｍＲＮＡ主要表达于弓状核，而ＳＨＲ表达量大于ＷＫＹ．ＰＰＤｍＲＮＡ表达于海马、下丘脑、中脑中央灰质、孤束核、胸髓．在齿状回、孤束核、内侧视前区，ＳＨＲＰＰＤｍＲＮＡ表达量少于ＷＫＹ；在弓状核、胸髓、海马ＣＡ１、ＣＡ２、ＣＡ３两者之间无差别．结论：ＳＨＲ弓状核ＰＯＭＣｍＲＮＡ增加和齿状核等ＰＰＤｍＲＮＡ减少可能与高血压的发病有关．

阿片肽与慢性单纯性苔藓瘙痒发生的相关性研究

目的初步探讨阿片肽对慢性单纯性苔藓瘙痒的生物学作用及相关研究。方法选择2020年4月—2021年3月于成都医学院第一附属医院皮肤科门诊治疗的慢性单纯性苔藓瘙痒患者分为2组,瘙痒组50例、无瘙痒组45例,另选取健康献血员的血浆(n=40)作为对照。采用放射免疫测定法测定标本的神经肽含量,用酶联免疫吸附试验(ELISA)测定β-内啡肽、亮啡肽和强啡肽的含量,受试者特征曲线(ROC)分析β-内啡肽、亮啡肽和强啡肽预测慢性单纯性苔藓瘙痒患者曲线下面积(AUC)。结果与正常对照组相比,无瘙痒组、瘙痒组患者血浆β-内啡肽、亮啡肽、强啡肽水平依次升高,差异具有统计学意义(P<0.05);与无瘙痒组相比,瘙痒组患者血浆β-内啡肽、亮啡肽、强啡肽水平升高,差异具有统计学意义(P<0.05)。与轻度瘙痒组相比,重度瘙痒组患者血浆β-内啡肽、亮啡肽、强啡肽水平较高,差异具有统计学意义(P<0.05)。β-内啡肽、亮啡肽和强啡肽联合检测对慢性单纯性苔藓瘙痒诊断价值优于单一检测,差异具有统计学差异(P<0.05)。结论联合检测β-内啡肽、亮啡肽、强啡肽水平对于慢性单纯性苔藓瘙痒患者有较高的诊断价值,为后续探究阿片肽对慢性单纯性苔藓瘙痒病情发展的影响机制提供了数据支撑。

左旋多巴诱发异动症大鼠模型纹状体神经元可塑性的研究

目的 研究左旋多巴诱发异动症 (LID)大鼠模型纹状体区FosB和特异性神经肽基因表达的变化 ,探讨LID时纹状体神经元的可塑性。方法 分别用免疫组织化学方法和逆转录聚合酶链反应技术 (RT PCR)检测大鼠纹状体区FosB和前脑啡肽原 (PPE)及前强啡肽原 (PDyn)基因的表达情况。用辣根过氧化物酶 (HRP)逆行示踪与免疫组织化学相结合的双重反应技术观测FosB的细胞分布状况。 结果 帕金森病 (PD)大鼠较正常大鼠损毁侧纹状体PPEmRNA表达明显增多而PDynmRNA表达减少。LID大鼠较PD大鼠PPEmRNA无明显增多 ,但PDynmRNA显著性增多。LID大鼠损毁侧纹状体区FosB阳性神经元明显增多 ,并且主要分布于纹状体黑质神经元内。结论 纹状体黑质神经元内即早基因FosB及其下游靶基因强啡肽的表达异常与大鼠LID的发生有关 ,表明LID的发生与直接通路的活动异常密切相关。

穴位按摩法对产妇强啡肽水平影响及镇痛作用的研究

目的:通过在第一产程活跃期时,对产妇实施穴位按摩,探讨此法对产妇体内强啡肽水平及分娩疼痛的影响。方法:将200例符合纳入标准的产妇随机分为两组,观察组100例,按中医理论对个体进行辨证后,采用穴位按摩法给予镇痛;另100例对照组,进行自然分娩。比较两组对产妇体内强啡肽水平及镇痛方面的影响。结果:穴位按摩后,观察组VAS评分低于按摩前(P<0.01);且观察组VAS评分低于对照组(P<0.01);分娩后,观察组静脉血强啡肽含量高于对照组(P<0.01)。结论:在第一产程活跃期进行穴位按摩可以增强产妇体内强啡肽水平,并减轻分娩疼痛。

强啡肽A及其受体拮抗剂对大鼠脊髓损伤后脊髓血流量的影响

目的：探讨强啡肽Ａ在脊髓损伤（ＳＣＩ）中的作用。方法：采用Ａｌｌｅｎ打击法复制大鼠ＳＣＩ模型，应用强啡肽Ａ放免检测技术和氢清除法分别测定伤段脊髓组织２４ｈ内强啡肽Ａ含量和脊髓血流量（ＳＣＢＦ）的变化，并观察脊髓蛛网膜下腔注射强啡肽Ａ及其受体拮抗剂ｎｏｒ－ＢＮＩ对ＳＣＢＦ的影响。结果：ＳＣＢＦ在伤后１０ｍｉｎ即有明显下降、２ｂ较１ｈ略有回升，４～８ｈ又进一步下降，两次下降相差显著；ＳＣＩ后脊髓蛛网膜下腔注射强啡肽Ａ可明显加剧脊髓缺血，而注射强啡肽受体拮抗剂ｎｏｒ－ＢＮＩ则可显著减轻脊髓缺血。脊髓强啡肽Ａ含量在伤后１０ｍｉｎ明显升高，１～２ｈ有所下降，但仍高于对照组，４，８ｈ较２ｈ略增加。强啡肽Ａ含量与ＳＣＢＦ的变化，两者呈显著相关性。结论：ＳＣＩ后强啡肽Ａ的过度释放是ＳＣＩ后继发性损伤的重要因素。

抗阿片肽血清对催产素增强电针镇痛的影响

本工作观察了大鼠侧脑室注射抗阿片肽血清对催产素增强电针镇痛作用的影响。脑室注射抗Beta-内啡肽血清(AEPS)使电针镇痛效应明显降低,但在注射催产素前预先注射AEPS对催产素增强电针镇痛作用无明显影响。注射抗强啡肽Al-13血清使电针镇痛明显降低,但却使催产素增强电针镇痛作用明显增强。无论抗甲-脑啡肽血清还是抗亮-脑啡肽血清对电针镇痛都无明显影响,对催产素增强电针镇痛的作用也不产生影响。上述结果说明,催产素增强电针镇痛的作用虽被脑内强啡肽Al-13部分对抗,但不受Beta-内啡肽和脑啡肽的影响,表明催产素增强针刺镇痛的作用不依赖于脑内内源性阿片肽系统。

c-fos基因的反义寡聚脱氧核苷酸鞘内注入减弱福尔马林引起的大鼠伤害性行为反应及脊髓背角Fos蛋白和强啡肽 A表达的研究(英文)

本研究通过鞘内注射 c-fos反义寡聚脱氧核苷酸 (antisense oligodeoxynucleotide,AS-ODN)封闭背角中 F os蛋白的表达 ,观察了 Fos蛋白合成和慢痛反应的关系 ,并分析 Fos蛋白合成和强啡肽 A(Dyn A)表达之间的关系。实验组动物 (n=5 )鞘内预先注射 c-fos的 AS-ODN(5 0μg,5μl) ,另两个对照组 (每组 n=5 )分别注射等量生理盐水和 AS-ODN的反序核苷酸 (reverseoligodeoxynucleotides,RS-ODN,5 0μg,5μl) ;4h后 ,各组动物均在一侧后肢脚掌皮下注射 formalin(5 % ,5 0μl) ,并立即用计算动物舔拭注射侧后脚掌累计时间的方法 ,检测大鼠的伤害性行为反应 ,行为检测后 1h处死动物 ,用免疫组化方法检查脊髓背角中 Fos蛋白阳性神经元的数量和强啡肽 A(1～ 8)的表达量。结果发现 ,和两个对照组相比 ,实验组动物 Formalin诱发的伤害性行为反应的第二相明显减弱 ,同时 Form alin注射侧背角 F os蛋白样免疫阳性细胞的数量和 Dyn A含量的灰度值明显减少。本研究结果提示 ,外周伤害性刺激引起的长期持续的痛反应是以 c-fos基因表达增加及受其调控的 Dyn A表达上调为基础的 ,由此推测在脊髓背角神经元中 Fos蛋白和 Dyn