Effect Of Celecoxib And Its Analogues On M Current And Pain Behavior

Pain is a complex physiological and psychological activity and one of themost common clinical symptoms, and pain is characterized by pain sensationand pain behavior response to the noxious stimuli. Pain can be caused byphysical and chemical stimuli such as mechanical stimulus, electric stimulus,high temperature, strong acid and alkali. All of these may lead to tissue injuryand increase sensitivity and excitability of nociceptors. TNF-α, NGF，IL-1β,BK，PGE2and other inflammatory mediators may enhance the sensitivity andexcitability of nociceptors through modulaion of the pain-relevent receptorsand ion channels. Pain signals are conducted through the unmyelinated C-fiberand myelinated A δ-fiber of spinal cord dorsal horn primary and secondarysensory neurons. In recent years, it has been a general interest to study themodulation of ion channels especially the potassium channels on thenociceptors.Potassium channel selectively allows the K+to pass through, and is themost distributed ion channel family that contains more than40members.Potassium channel plays important role in regulating cell excitability,maintaining the resting membrane potential and regulation of apoptosis etc.KCNQ gene family is one of important subfamily of voltage-gated potassiumchannel. There are five members within the KCNQ family, KCNQ1-5, alsoknown as Kv7.1-7.5. Heteromers of two KCNQ members, KCNQ2andKCNQ3, may constitute the molecular basis of the M potassium channel. Mcurrent is a voltage and time-dependent, low threshold, slow activation, slowdeactivation and non-inactivating outward K+current. It is named the "Mcurrent" because of its inhibition by excited M-type cholinergic receptor. Thelow-threshold (-60mV) activation of M current makes the current to be thekey fact in determining the neuronal resting membrane potential and excitability. M channels are distributed in various types of cells and tissues,including sympathetic ganglia, dorsal root ganglia, hippocampus cells andother central nervous system cells. It was proved that M current expressed indorsal root ganglia play an important role in nociception. Because of theimportance of M-channel in pain, more and more researches are currentlyfocused on studying the pharmacological augmentation of K+channels inneurons as a means to reduce their excitabilityCelecoxib, a COX-2specific inhibitor, belongs to non-steroidalanti-inflammatory drug. Celecoxib is usually used to treat inflammatory painand regulate function of immune system etc, through reducing the generationof PGs. In our previous study, we found that celecoxib can activate most of theKCNQ channel including KCNQ2/3heteromers and native neuronal Mcurrent. We demonstrated that this activation was a direct effect on the channelrather than through the inhibition of COX-2.Based on the importance of M-current in pain and the stimulatory effectsof celecoxb on M-current, we hypothesize that, in addition to reduce thegeneration of PGs by inhibiting COX-2, activation of M current in DRGneurons may also be involved in the analgesic effect of celecoxib as aNSAIDs.Objective: In this study, we use celecoxib (CXB) and its structuralanalogues UM-celecoxib (UMC) or DM-celecoxib (DMC）as tool drugs(UMC has a higher COX-2inhibitory activity but DMC does not inhibit theactivity of COX-2), to observe the effect of these drugs on KCNQ2/3currentexpressed in HEK293cells and M current in DRG neurons, and on the painbehaviors induced by BK, mechanical and thermal stimuli, and to investigatethe effect of these three drugs on hyperalgesia of neuropathic pain. Then wewill analyze the relationship of M current modulation by these drugs and theireffects on pain behavior in order to explore the role of M current modulationby celecoxib during pain therapy.Methods:1, Electrophysiological experiments: Peforated patch clamptechnique was used to study the effect of CXB, DMC and UMC on M current KCNQ2/3current expressed in HEK293cells and M current from DRGneurons.2, Behavioral studies:(1) Bradykinin-induced acute spontaneous pain.The right hind paw of the animal received an intraplantar injection BK and/orCXB,UMC, DMC and the nocifensive responses (licking, biting, lifting andflinching) were recorded using a video camera for30min. The videos wereanalyzed by an observer unaware of the treatment allocations.(2) Mechanicalwithdrawal thresholds were measured using calibrated von Frey filamentsapplied to the plantar surface of the paw.(3) To test for thermal hyperalgesia,radiant heat was applied to the plantar surface of a hind paw from underneatha glass floor using a ray of light from a high-intensity lamp bulb. The pawwithdrawal latency was recorded automatically when the paw was withdrawnfrom the light.3, The chronic constriction injury (CCI) neuropathic painmodel was used to test the effect of CXB, UMC, DMC on hyperalgesia ofneuropathic pain.Results：一、Electrophysiological experiments1CXB, DMC, UMC significantly increase the KCNQ2/3current expressed inHEK293cells with the efficiency of DMC> CXB> UMC.2CXB, DMC， UMC concentration-dependently increase the KCNQ2/3current expressed in HEK293cells. EC50for DMC, CXB and UMC are2.5±0.2μM,4.5±0.7μM, and22.7±3.0μM, respectively.3CXB，DMC，UMC increase the M current from DRG neurons with lowersensitivity and efficiency than that in HEK293cells but with similar order ofDMC>CXB>UMC.二、Behavioral studies1CXB, DMC, UMC significantly attenuate the inflammatory pain induced byBK. BK-induced nocicepfensive time in control group is137±18.9s, CXB+BK group is65±11.8s, P≤0.01vs control, DMC+BK group is82±12.1s, P≤0.05vs control, UMC+BK group is62±13.2s,P≤0.01vs control.2CXB, DMC, UMC significantly antagonize the nocifensive response tomechanical stimulai. The mechanical withdrawal threshold are21±5.9g in solvent control group,42±7.1g in CXB group(P≤0.01vs control),45±7.4g inDMC group(P≤0.05vs control) and45±7.2g in UMC group (P≤0.05vscontrol)；56±4.3g in positive RTG control group(P≤0.001vs control). Thereare no significant differences between each drug group.3CXB, DMC, UMC significantly antagonize the thermal pain behavior. Thepaw withdrawal latency are14±1.4s in the solvent control group，20±2.0s inCXB group(control P≤0.05)；24±1.8s in DMC group(P≤0.001vs control)；24±1.2s in UMC group (P≤0.001vs control)；27±1.2s in positive controlRTG group(P≤0.001vs control) There are no significant differences betweeneach drug group.4CXB and DMC relieve hyperalgesia of neuropathic pain in rat chronicconstruction injury model.The basic mechanical pain threshold and thermal pain threshold in eachgroup are not significantly different before operation. The basic mechanicalpain threshold is: control25±3.7g，n=8；CXB21±2.2g，n=8；DMC25±3.9g，n=8；RTG21±1.5g，n=8; and the basic thermal pain threshold is: control23±1.7s，n=8；CXB24±1.3s，n=8；DMC24±1.8s，n=8；RTG23±0.7s，n=8. In the first day after surgery, the mechanical pain threshold and thermalpain threshold are significantly decreased. The mechanical pain threshold is:contro12±1.8g，n=8；CXB14±1.0g，n=8；DMC11±0.4g，n=8；RTG10±0.4g，n=8）; and the thermal pain threshold is: control17±1.3s，n=8；CXB21±0.8s，n=8； DMC18±0.7s，n=8；RTG17±0.9s，n=8.In the fifth day after surgery, mechanical pain threshold in drug groups issignificant different compared with the solvent control (CXB7.4±0.2g，n=8；DMC7.0±0.2g，n=8；RTG8.1±0.4g，n=8; vs control4.3±0.3g，n=8, P≤0.01).On the other hand, thermal pain threshold（control18±1.0s，n=8；CXB20±1.1s，n=8；DMC19±1.0s，n=8；RTG17±1.6s，n=8） is not significantlydifferent between all groups.In the tenth day after surgery, mechanical pain threshold in drug groups issignificant different compared with the solvent control,(control group4.1±0.2g CXB group8.5±0.5g，DMC group7.0±0.7g, RTG group6.6±0.5g; P≤0.01vs control group). For the thermal pain threshold, only CXB grouphas significantly higher threshold compared with the solvent control group(solvent16±1.1s；CXB21±1.7s, P <0.05).In the fourteenth day after surgery, all drug-treated groups havesignificant higher mechanical and thermal pain threshold compared with thesolvent control group. Mechanical pain threshold (control4.0±0.2g；CXB5.4±0.2g；DMC5.2±0.5g；RTG4.6±0.3g; for all drug groups, P <0.01compared with the control group). Thermal pain threshold (control16±0.9s；CXB20±1.3s；DMC20±1.2s；RTG19±0.7s; for all drug groups, P <0.05compared with the control group）.Conclusion: This study demonstrates that1) CXB and its analogues canconcentration-dependently enhance the current amplitude of KCNQ/Mchannel expressed in HEK293cells and in DRG neurons. The effects of CXBand its analogues do not depend on the inhibition of COX-2.2) CXB and itsanalogues can attenuate the nociceptive response caused by a variety ofnoxious stimuli.3) CXB and its analogues can relieve hyperalgesia inneuropathic pain. In summary, apart from inhibition of COX-2, activation ofthe M current may also be involved in the analgesic mechanism of CXB.