Effect And Mechanism Of ARA290 Pretreatment On The Neuronal Response And Mechanical Hypersensitivity Induced By Capsaicin

ARA 290 is a polypeptide that is derived from the receptor-binding domain Helix B in erythropoietin.It consists 11 amino acids and does not retain the erythropoietic function.ARA 290 has been reported to be beneficial in many clinical applications such as diabetes and sarcoidosis.ARA 290 is also able to protect tissue from injury and repair tissue post-injury.This is probably due to the property of ARA 290 to specifically target the innate repair receptor(IRR)and down-regulate inflammation at the injury site.Remarkably,apart from being a conventional drug that has many side effects in clinic,such as the recombinant human erythropoietin(rhEPO),ARA 290 is generally considered safe when evaluated in the animal toxicity and the human patient studies.The short half-life time of ARA 290,that minimizes the exposure of tissues to high concentration of drug,might be the contributing factor.Particularly,recent studies suggest that ARA 290 is able to improve the neuropathic symptoms and relieve neuropathic pain.Neuropathic pain is the condition that pain fibers in the somatosensory system are affected due to diseases or tissue lesions,with usual symptoms including hyperalgesia and allodynia.Hyperalgesia is characterized by the elevated pain sensitivity,and allodynia is characterized by the pain response to normally non-painful stimuli.Common diseases that could cause neuropathic pain include spinal cord injury,multiple sclerosis,and diabetes,as well as cancer and chemotherapy used to treat cancer.In the peripheral somatosensory system,neuropathic pain is usually caused by sensitized pain fibers developing abnormal spontaneous post-damage activity.In the central nervous system,the mechanism is more complicated,which could in-volve increased sensitivity of the spinal neurons by phosphorylation of the N-methyl-D-aspartate(NMDA)subunits,and the declination of inhibitory effects of interneurons.Besides those abnormalities in the neural system,inflammation also plays an important role in raising and maintaining neuropathic pain.Typically ARA 290 is believed to alleviate neuropathic pain by suppressing inflammation through targeting the Innate Repair Receptor(IRR),however,whether it has direct analgesic effect on the central and peripheral nervous system remains unknown.Recently,the immune system has been shown to have many implications in pain transmission,and some immune molecules could directly activate peripheral pain fibers.These recent findings encourage us to speculate that ARA 290 might have direct effects on transient receptor protein channels,such as TPRV1-4,TPRM8 and TRPA1,which are responsible for most of the pain sensing.In the peripheral nervous system,TRPV1 is mainly located in the dorsal root ganglion(DRG)of the spinal cord and small and medium-sized neurons called nociceptors in the trigeminal ganglion(TG).These small-diameter neurons are mostly connected to fine myelinated A? fibers or unmyelinated C fibers,and are therefore considered to be closely related to pain transmission.TRPV1 specifically binds to capsaicin.When activated,it preferentially allows calcium ions to pass through its ion channel,causing an increase in intracellular calcium concentration,and an increase in calcium ion concentration to cause substance P(SP)and calcitonin gene-related peptide(CGRP)and other neuropeptides and excitatory amino acid release,which in turn produces pain.Based on the above studies,we propose whether the immune medium ARA290 directly targets peripheral nociceptors by blocking or affecting pain receptors,ie whether ARA290 exerts an analgesic effect by directly acting on the neuronal TRPV1 receptor.Therefore,in this study,we used the capsaicin induction model of DRG neurons and TG neurons to investigate whether the pretreatment of ARA290 in the immune medium reduced capsaicin-induced DRG neurons and TG neurons by using calcium imaging,cell culture and behavioral tests.The response,and whether this effect is mediated through the TRPV1 receptor.PART ? ARA290 PEPTIDE PRETREATMENT REDUCES CAPSAICIN-INDUCED NEURONAL RESPONSES BY INCREASING THE TRPV1 RECEPTOR RESPONSE THRESHOLDObjectives:To investigate whether the pretreatment of ARA290 in the immune medium reduces the response of capsaicin-induced dorsal root neurons and trigeminal neurons,and whether this effect is mediated through the TRPV1 receptor.Methods:The capsaicin induction model using DRG neurons and TG neurons was studied by using calcium imaging and cell culture.The specific experimental methods are as follows:1.Dissect and isolate the neuronal cells in the dorsal root ganglia(DRG)and trigeminal ganglion(TG)of mice,stained with calcium ion indicator Fluo-4 AM staining solution,and measured by laser confocal microscopy.Fluorescence signal intensity of neurons for various stimuli,including ARA290,capsaicin,and KCl solutions.100 DRG neurons and TG neurons were divided into four groups,namely Cap group,Cap+A group,KCl group,and KCl+A group,with 25 cells in each group.Cells in Cap+A and KCl+A groups:3 nM ARA 290 was pretreated,followed by 3 ?M Capsaicin and 50 mM KCl;Cap and KCl were not pretreated,and 3 ?M Capsaicin and 50 mM KCl were added,respectively.2.Using the isolated DRG neurons to test the agonist of the temperature-sensing receptor,we recorded each stimulated calcium ion in 10 neuronal cells in both cases with agonist alone and 3 nM ARA290 simultaneously.Signal response.Agonists include:agonist of TRPM5 100 ?M menthol,agonist of TRPA1 10?Mallyl isothiocyanate(AITC),agonist of TRPV2,3 100 ?M 2-APB,agonist of TPRV41 ?M GSK1016790A3.Separate DRG and TG neurons were stained with fluo-4 AM,and then different concentrations of Capsaicin solution were applied at concentrations ranging from 0.1 ?M to 10 ?M.The same concentration of capsaicin solution was then mixed with three different concentrations of ARA290(0.3 nM,1 nM and 10 nM)solutions and applied to other groups of DRG neurons and TG neuronal cell solutions.Based on the values of homogenization,we plotted the concentration curve of the effect of ARA290 on Capsaicin.4.The TRPV1 channel gene was overexpressed in HEK293 cells,and the cells expressing the TRPV1 protein also expressed the mCherry protein as a reporter gene.We stained the cells with fluo-4 AM and tested their response to calcium signals induced by 10 ?M Capsaicin and 3 nM ARA 290.Results:1.ARA 290 specifically inhibited capsaicin-evoked responses in neurons of the dorsal root ganglion(DRG)and the trigeminal ganglion(TG):Single DRG neurons and TG neurons showed a calcium ion response signal to 50 mM KCl and 3 ?M Capsaicin,but the addition of ARA290 specifically inhibited the capsaicin-induced calcium ion response of DRG neurons to TG neurons.When the calcium ions of DRC neurons induced by capsaicin in multiple sample neurons were detected,it was also found that ARA 290 pretreatment specifically inhibited the calcium ion response of Capsaicin-induced DRG neurons and TG neurons.However,the calcium signal response caused by KCI depolarization was not significantly inhibited in dorsal root neurons and trigeminal neurons,either in a single neuron or after increasing the sample size.Therefore,it is speculated that ARA 290 may specifically bind to the TPRV1 channel Capsaicin receptor.2.ARA290 specifically targets binding to the TRPV1 channel to reduce the capsaicin response:It was not found in the experiment that ARA290 can significantly inhibit or enhance the response of any agonist challenge,indicating that we found that ARA290 can specifically inhibit TRPV1 channels without affecting other thermal receptors.3.ARA290 increases the threshold for the response of the TRPV1 channel to capsaicin:It was observed in both DRG and TG neurons that the initial concentrationcurve of Capsaicin shifted to the right after ARA290 was applied,and it also increased the threshold of response of Capsilin to TRPV1 channel,and this effect was dose-dependent,high concentration.ARA290 will raise the threshold higher.At the same time,the EC50 value of Capsaicin became larger in the presence of ARA 290 compared to Capsaicin alone,suggesting that ARA 290 may be a competitive antagonist of Capsaicin.4.ARA290 can inhibit Capsaicin reaction in a dose-dependent manner:10 ?M Capsaicin caused a strong calcium response in HEK293 cells,while applying 10 ?M Capsaicin to the cells while adding different concentrations of ARA290 solution(from 0.1 nM to 10 nM),it was found that ARA290 can inhibit the Casseline-induced calcium signal response.And this inhibition exhibited a concentration-dependent,half-maximal inhibitory effect concentration(IC50)of approximately 1 nM.This indicates that ARA290 inhibits the Capsaicin-evoked calcium ion signaling response of TRPV1 overexpressing HEK293 cells in a dose-dependent manner.Conclusion:1.In a single DRG neuron and TG neuronal cell,ARA 290 specifically inhibits the TRPV1 channel-mediated neuronal response induced by capsaicin,which is independent of KC1-induced TRPV1 depolarization,and other Heat receptor independent;2.In multi-neuronal cell assays and HEK293 cell lines,ARA 290 was able to specifically inhibit the capsaicin-induced excitation of TRPV1 channels,and this inhibition was dose-dependent;3.The presence of ARA290 can increase the response threshold of TRPV1 channel,and there is competitive antagonism between ARA290 and capsaicin,which is an antagonist of TRPV1.Taken together,our study suggests that ARA 290 may be a novel antagonist of the TRPV1 channel.PART ? ARA290 ALLEVIATES NEUROPATHIC PAIN BY TARGETING TRPV1 CHANNELSObjectives:The capsaicin-induced mouse pain model was used to observe whether the immune medium ARA290 can alleviate pain and hyperalgesia,and whether this relief is dose-dependent.Methods:1.Paw withdrawal threshold assay:Three groups of 8 mice were injected s.c.with 1 nM ARA 290,10 nM ARA 290 or PBS only in the plantar region of the left hind paw.After 15 min,these mice were injected with 10 ?M capsaicin solutionat the same site of ARA 290 injection and returned to observation cages.For the control group(n=8 mice),mice were injected with 10 ?L saline solely and returned to observation cages.After 30 min of resting,the paw withdrawal threshold was measured using the Ugo Basile analgesimeter.The force of von Frey filament applied to the ventral surface of the hind paw was increased gradually until the paw withdrawal threshold was reached.Measurements were repeated two times and mean was used in analysis.2.Paw withdrawal frequency assay:Mice were injected with 10 ?M capsaicin solutionor saline only in the plantar region of the left hind paw.After 15 min,mice injected with capsaicin were divided into four groups(n=8 mice for each group),while three groups of mice were then injected with 1 nM ARA 290,10 nM ARA 290 or PBS only in the same region of the paw.Mice then were put in a plastic chamber to rest and the paw withdrawal frequencies were measured at 1,2,4,8,24 hr post capsaicin injection.The von Frey filament was applied to the plantar surface of the hind paw and the paw withdrawal frequency was then measured.The tests were repeated 10 times and the percentages of positive responses were used in statistical analysis.Results:1.ARA290 inhibits the pressure threshold of capsaicin-induced paw withdrawal reflex in living mice:Injection of capsaicin is a hallmark of mechanical hyperalgesia.Injecting PBS alone did not increase the threshold for reflexive reflex.In contrast,the application of 1 nM and 10 nM ARA 290 significantly increased the threshold for degenerative reflex,and this effect was dose-dependent,1 nM ARA 290 The treatment only slightly increased the reflection threshold,while 10 nM ARA 290 almost completely inhibited the reduction of capsaicin's threshold for the reduction of the foot.2.ARA290 inhibits the frequency of capsaicin-induced contraction reflex in living mice:The hyperalgesia induced by capsaicin can be partially relieved by ARA290,but not by PBS,and this alleviation is also dose dependent.Conclusion:The results of the in vivo mouse capsaicin induction model indicate that ARA290 can alleviate capsaicin-induced mechanical hypersensitivity by antagonizing the TRPV1 channel.