| ObjectivesPain is the most common syndrome in patients,and it can severely impact the quality of their lives.The mechanism study of formulation of pain has been developing due to the establishments of pain animal models recently.Nowadays,these studies have been showed that volted-gated sodium channels play some no negligible roles in the path of pain transmission.These factors including dysfunctions,different kinds of sodium channel subtypes and disorder of and expression,are the culprit in emerging of pain,and the most influsive fact,the tetrodotoxin-resistant(TTX-R)sodium channel Nav1.8 became the study focus of pain.BmK I is a single component from BmK(Buthus martensii Karsch)toxin.It can be used to establish the animal model which has the feature of short-lasting time and one-way behavior curse to simulate spontaneous pain.And the BmK I pain model has the appearance of breaking out in pain that made it a tool with which the breakout of pain can be studied clinically.The study of mechanisms in bone cancer pain developed due to the animal model establishment with walker256 mammary gland carcinoma cells in rats.Recently research finds that bulleyaconitine A can release pain significantly in clinical because of its capability in regulating the electrophysiological properties of sodium channel Nav1.8.So,is there any relationship between Nav1.8 and appearance of pain or relief of pain.In this study,expression and distribution of voltage gated sodium channels were systematically studied through behavior test,real-time PCR,immunohistochemical technologies in rat DRG neurons.Thus the relationship between pain and sodium channel should been discussed.The sodium current of Nav1.8 in bath solution of 10 ?M BLA was recorded by whole-cell path clamp.The mechanisms of Nav1.8-mediated pain signal transmission of rats will be studied Methods1.Mechanism of Nav1.8 in BmK I scorpion sting pain model12 SD(Sprague-Dawley)rats were divided into 2 groups: Model of Bm K I(B group,n=6),control group(control,n=6).BmK I(10?g/50?l)was hypodermic injected at the left metapedes of rats in B group to establish the model of BmK I model.To investigate the spontaneous pain 2h after the injection,the long-time mechanical allodynia and thermal hyperalgesia 4h and 8h after the injection.The expression and location of relative mRNA and protein of Nav1.8 were examined by real-time PCR and immune-blotting respectively.2.Mechanism of Nav1.8 in bone cancer pain model30 female Wister rats were divided into 3 groups: Model of bone cancer pain(Walker256,n=10),sham-operated group(sham,n=10),control group(control,n=10),Walker 256 mammary gland carcinoma cells were injected into the tibia medullary cavity.The weight,free walk behavior score,threshold of mechanical hyperalgesia and threshold of thermal stimulation were recorded,and the image science examination and pathological examination were taken.3.Regulation Effect of Bulleyaconitine A on Sodium Channel Nav1.8The DRG cell was acute isolated and divided into two groups(control n=6,and BLA group n=6),the sodium current of Nav1.8 was recorded by whole-cell patch-clamp in extracellular fluid with 500nmol/L TTX.The DRG cell was clamped in-60 mV,and those in BLA group was in bath solution with 10?M BLA.Results1.Mechanism of Nav1.8 in BmK I scorpion sting pain modelThis study exhibited that: Intraplantarinjection(i.p.)of BmK I(10 ug)(1)can induce spontaneous pain for up to 2 h,long-time mechanical allodynia,and thermalhyperalgesia in the injection side;(2)Relative mRNA and protein of Nav1.8 were examined by real-time PCR and immune-blotting,respectively,which exhibited both up regulated at different time courses in Bmk I treated groups.(3)Compared with na?ve groups,co-labeled rate of NF200,CGRP,or IB4 with Nav1.8 was changed in time courses by double immune-fluorescent histochemistry.2.Mechanism of Nav1.8 in bone cancer pain modelThe weight of rats decreased in W group.The results of imaging examination demonstrated the upper tibia bone destruction at 21 days after operation.The results of pathological examination demonstrated the hyperplasia and destruction of bone,tumor growth,a large number of tumor cells in tibia.Free walk behavior score of W group increased at D12 instead of declining,and the differences between W group and other groups were significant(P<0.05)at D16 and D20.The threshold of mechanical hyperalgesia of W group was lower than those of C group from D4 to D20,the differences were significant(P<0.05).The threshold of mechanical hyperalgesia of S group was lower than those of C group from D4 to D12,the differences were significant(P<0.05).Threshold of thermal stimulation of W group was lower than those of C group from D4,the differences were significant(P<0.05).Threshold of thermal stimulation of S group was lower than those of C group only at D4 and D8,the differences were significant(P<0.05).Compared with other groups,The mRNA expression levels of Nav1.8(W :134.95±4.24)in DRG were up-regulated significantly at the operation side of W group(P<0.05).3.Regulation Effect of Bulleyaconitine A on Sodium Channel Nav1.8In bath solution with 10?M BLA,peak sodium current of Nav1.8 was declined at steady-state activation,the differences were significant(P<0.05)while it was not changed at steady-state inactivation.And the IV curve and GV curve were shifted to depolarization in steady-state activation and the GV curve was shifted to depolarization too in steady-state inactivation but the differences were not significant(P?0.05).The current of Nav1.8 declined after bathing with 10?M BLA,and Km of the curve was not changed.ConclusionsThese results above demonstrated that Nav1.8 play important role in induction and maintain stages of BmK I inflammatory pain model.In addition,differential types of DRG neurons take differential patterns in regulating of Nav1.8.All these researches moved forward to understand the mechanisms of Nav1.8 targeted in BmK I scorpion sting pain model.Injection of Walker 256 mammary gland carcinoma cells in the tibia medullary cavity was associated with bone cancer pain obviously.The expression of Nav1.8 mRNA was up-regulated significantly in DRG,associated with bone cancer pain.The expression level in DRG indicated that the change of small neuron was more important in the emerging of bone cancer pain.Bulleyaconitine A can regulate the peak sodium current of Nav1.8 at the concentration of 10?M,and the effect was appeared after 10 min in bath solution.There was no other effect of Bulleyaconitine A on the electrophysiological properties of Nav1.8. |
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