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.

Research review Neuropeptides and traumatic brain injury

It has been proved that endogenous opioids play an important pathophysiological role inthe lesions secondary to central nervous system trauma.Opiate antagonists and opioid antisera havebeen found to improve the outcome of experimental brain injury,Thyrotropin-releasing hormone(TRH),which appears to act in part as a functional antagonist of opioid system,has also beenproved to be effective in the treatment of experimental brain injury.The developments of these as-pects in our laboratory are reviewed.

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.

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.

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.

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

Plasma dynorphin A1-13 levels were measured in 33 patients with mitral stenosis before and after percutaneous balloon mitral commissurotomy (PBMC). The results show that the basal levels of plasma dynorphin in blood from the antecubital vein in the patients were significantly higher than those in 31 healthy control subjects. The increase in circulating dynorphin closely correlated with the functional cardiac status and the presence of atrial fibrillation. Ten to fifteen minutes after PBMC, plasma dynorphin levels in blood from the femoral vein increased significantly. Seventy-two hours after the procedure, the levels of plasma dynorphin in blood from the antecubital vein had decreased significantly , but they did not decrease to the normal range. Plasma dynorphin levels in blood from the femoral vein were positively correlated with the mean left atrial pressure and the mean right atrial pressure before the first balloon inflation. Plasma dynorphin levels in blood from the antecubital vein were positively correlated with the heart rate and the mean transmitral pressure gradient, and negatively with the mitral valve area before and 72 hours after PBMC.

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

The effect of intrathecal injection of dynorphin A (1-17) on second messenger systems of spinal cord relative to behavioral change in rats was studied. Dynorphin A (1-17) 5 ,10 (20nmol) caused dose-dependent flaccid paralysis of hindlimbs. Dynorphin A (1-17) 10, 20 nmol dose-dependently decreased spinal adenylate cyclase (AC) activity, cyclic AMP production, calmodulin (CaM) level and cyclic-nucleotide phosphodiesterase(PDE)activity 10 min after intrathecal injection. They recovered to a varying extent two hours later. Pretreatment with selective κ-opioid receptor antagonist nor-BNI 30 nmol 10 min before dynorphin A (1-17) markedly antagonized the effects of dynorphin A (1-17 ) at 20 nmol on hindlimb paralysis and inhibition of intracellular second messengers. The L-type calcium channel blocker verapamil (100nmol) also played a role in blocking dynorphin neurotoxicity. The NMDA receptor antagonist APV could partially or completely block dynorphin inhibition of CaM level and PDE activity without affecting paralysis and decrease of AC-cAMP level induced by dynorphin A(1-17) 10 min after intrathecal injection.

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.

Analgesic effect of different moxibustion durations in rheumatoid arthritis rats

OBJECTIVE:To observe the influence of different moxibustion durations on hypothalamic pro-opiomelanocortin(POMC)and prodynorphin(PDYN)mRNA expressions and plasmaβ-endorphin(β-EP)content in rheumatoid arthritis(RA)rats,to understand the mechanism of moxibustion analgesia and its dose-effect relationship.METHODS:Twelve male Wistar rats were randomly selected from 48 male Wistar rats as a normal control group.The RA model was created by raising rats in a windy(blowing with electric fan),cold(6℃±2℃),and wet(80%-90%humidity)environment for 20 days,12 h each day.This was followed byinjectionofFreund'scompleteadjuvant(0.15 mL)into the ankle.Then,rats were randomly divided into a model group,moxibustion groupⅠ,and moxibustion groupⅡ,with 12 rats in each group.In moxibustion groupsⅠandⅡ,moxibustion was given at Shenshu(BL 23)and Zusanli(ST 36)for 20and 40 min,respectively,once daily for 15 days.Hypothalamic POMC and PDYN mRNA expression levels and plasmaβ-EP content were determined.RESULTS:Compared with the normal group,the pressure pain threshold decreased,while the hypothalamic POMC and PDYN mRNA expression levels and plasmaβ-EP content increased in the moxibustion groups(P<0.01).Compared with the model group,the pressure pain threshold,hypothalamic POMC and PDYN mRNA expression levels and plasmaβ-EP content in the moxibustion groups increased significantly(P<0.01).Compared the moxibustion groupⅠ,the pain threshold,hypothalamic POMC and PDYN mRNA expression levels and plasmaβ-EP content in moxibustion groupⅡsignificantly increased(P<0.01).CONCLUSION:Moxibustion has an analgesic effect and increases hypothalamic POMC and PDYN mRNA expression levels and plasmaβ-EP content inRArats.TheanalgesiceffectinmoxibustiongroupⅡisbetterthanthatinmoxibustiongroupⅠ.

Involvement of Opioid Peptides in the Analgesic Effect of Spinal Cord Stimulation in a Rat Model of Neuropathic Pain

Spinal cord stimulation(SCS)-induced analgesia was characterized,and its underlying mechanisms were examined in a spared nerve injury model of neuropathic pain in rats.The analgesic effect of SCS with moderate mechanical hypersensitivity was increased with increasing stimulation intensity between the 20%and 80%motor thresholds.Various frequencies(2,15,50,100,10000 Hz,and 2/100 Hz dense-dispersed)of SCS were similarly effective.SCS-induced analgesia was maintained without tolerance within 24 h of continuous stimulation.SCS at 2 Hz significantly increased methionine enkephalin content in the cerebrospinal fluid.The analgesic effect of 2 Hz was abolished byμorκopioid receptor antagonist.The effect of 100 Hz was prevented by aκantagonist,and that of 10 kHz was blocked by any of theμ,δ,orκreceptor antagonists,suggesting that the analgesic effect of SCS at different frequencies is mediated by different endorphins and opioid receptors.

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

Acupuncture-related techniques for the treatment of opiate addiction:a case of translational medicine

Drug addiction is a chronic brain disorder characterized by withdrawal symptoms that occur during drug abstinence and a high tendency of relapse.Compared with the currently available pharmacological interventions,acupuncture therapy has the potential to help drug addicts stay away from drugs without major adverse side effects.It has taken decades of research to optimize the parameters of electrical acupoint stimulation for detoxification and for relapse prevention,as well as to establish a safe and easy procedure by which drug addicts can use it on themselves.The discovery that acupuncture can trigger the release of opioid substances from the brain in the 1970s provided the inspiration.Following this,basic research on animals made it possible to understand the mechanisms of action and establish the procedure for treating drug addictions.This article reviews the past,present,and foreseeable future regarding the use of acupuncture-related technique for the treatment of opiate addiction from the perspective of translational medicine.

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.