Upregulation Of TRPA1 In The Spinal Cord Contributes To The Development And Maintenance Of Neuropathic Pain Via The CaMK2-CREB Cascade

BackgroundNeuropathic pain（NP）is a direct pain caused by a lesion or disease of the somatosensory nervous system,which is the consequence of a complex interplay of mechanisms in the peripheral and central nervous systems that are among the most intractable of pain syndromes.Transient receptor potential channel（TRP）is a common ion channel in mammalian tissues,which is widely distributed in the central and peripheral nervous systems and involves the perception of temperature,toxic substances and pain.It plays an important role in immune response and nociceptive perception.Transient receptor potential cation channel,subfamily A,member l（TRPA1）is a non-selective calcium permeability cation channel of the TRP channel superfamily,and it is a stimulant sensor for many compounds.It plays an important role in the transmission and integration of nociceptive signals.Previous studies have shown that TRPA1 is highly expressed in the terminals of primary sensory neurons,mediate the release of Ca²⁺in the cytoplasm of neurons,enhance the excitability of neurons and mediate nociception at the most upstream of pain signals.It is also present in spinal dorsal horn neurons and some non-neural tissue cells.It has been reported that spinal cord level TRPA1 is involved in maintaining neuropathic pain caused by nerve root constriction model,but the specific mechanism remains unclear.Calcium/calmodulin dependent protein kinase 2（CaMK2）is a multifunctional serine/threonine protein kinase.The increase of intracellular Ca²⁺concentration can stimulate CaMK2 phosphorylation.Activated phosphorylated CaMK2 is involved in the sensory processing of various nociceptive stimuli,including nociceptive stimuli in spinal cord transmission.Cyclic adenosine monophosphate response element binding protein（CREB）is a nuclear transcription factor that stimulates gene transcription.Activated CaMK2 can phosphorylate CREB,to increase CREB transcription activity,and then regulate the transcription of target genes,which is involved in the formation of pain persistence and hyperalgesia.Previous studies have found that when the TRPA1 activator AITC activates the TRPA1 ion channel in cardiomyocytes,the intracellular Ca²⁺peak dose-dependently increases and activates the CaMK2-dependent signaling pathway,which ultimately enhances the contractile function of cardiomyocytes.However,whether TRPA1plays a role in neuropathic pain caused by Spared nerve injury（SNI）in rats and whether TRPA1 participates in the development of neuropathic pain through CaMK2-CREB pathway has not been reported yet.ObjectiveIn this study,the Spared nerve injury（SNI）rat model was used to observe the expression of TRPA1 in the lumbar spinal cord and the specific distribution of cell types,and to demonstrate the role of TRPA1 in the development of neuropathic pain.Further exploration of the possible downstream molecular mechanism of TRPA1involvement in neuropathic pain caused by SNI model,whether TRPA1 activates CaMK2-CREB signaling pathway mediates neuropathic pain.Methods1 Expression of TRPA1 and distribution of cell types in spinal cord lumbar enlargement after Spared nerve injury in ratsTo observe the SNI rat pain behavior and postoperative the expression of TRPA1in spinal lumbar enlargement,male SD rats were randomly divided into surgery group（SNI group）and sham operation group（Sham group）（n=15）before surgery,Rats were assessed for Paw withdrawal threshold（PWT）at 3 days,1 day,and 1,3,5,7,9,12,and 14 days after surgery.Each group three rats were taken at 0,3,7 and 14 days after surgery,the expression level of TRPA1 in the lumbar spinal cord was measured by Western Blot.After the completion of the pain test,the spinal cord lumbar enlargement was performed 14 days after SNI.The immunofluorescence double-labeling technique was used to detect the relationship between TRPA1 and neuronal marker NeuN,astrocyte marker GFAP and microglia marker IBA1,to observe the type of cells in which TRPA1 is expressed in the spinal cord.2 The effect of intrathecal injection of TRPA1 inhibitor HC-030031 on neuropathic pain behavior of Spared nerve injury model in rats（1）To observe the effect of pre-administration of HC-030031 on the occurrence of neuropathic pain,successful rats with intrathecal catheterization were randomly divided into 6 groups（n=9）:Sham+Vehicle group,SNI+Vehicle group,SNI+HC-030031 10μg group,SNI+HC-030031 50μg group,SNI+HC-030031 100μg group and HC-030031 100μg group,intrathecal injection of TRPA1 inhibitor HC-030031 or Vehicle pretreatment 30 minutes before surgery,once a day to postoperative 7 days.The PWT of rats was measured before surgery3,1 day,and 1,3,5,7,9,12,and 14days after surgery.The spinal cord lumbar enlargement was performed 2 hours after the last administration,and the expression levels of CaMK2 and CREB were detected by Western Blot.（2）To observe the effect of HC-030031 on established neuropathic pain,successful rats with intrathecal catheterization were randomly divided into 3 groups（n=6）:Sham+Vehicle group,SNI+Vehicle group and SNI+HC-030031.Intrathecal injection of HC-030031 or Vehicle was started on the 7th day after surgery（once every day for 5 days）,and the PWT was measured 3 days,1 day before operation,and 1,3,5,7,9,11,and 14 days after operation.Vehicle:10%DMSO.3 Effects of TRPA1 activator AITC on behavior in rats（1）To observe the effect of AITC activation of peripheral nerve TRPA1 on behavior in rats,male SD rats were randomly divided into 3 groups（n=6）:Vehicle group,AITC 100μg group and AITC 300μg group,TRPA1 activator AITC or Vehicle was injected subcutaneously into the left plantar in rats,the PWT was measured 3days,1 day before injection,and 0.5h,1h,2h,1,3,5,and 7 days after injection.（2）To observe the effect of pre-insertion of TRPA1 on the behavior of peripherally activated TRPA1,rats with successful intrathecal catheterization were randomly divided into 2 groups（n=6）:Vehicle+AITC 300μg group and HC-030031100μg+AITC 300μg Group,AITC 100μg or Vehicle was injected intrathecally 30minutes before subcutaneous injection of HC-030031 300μg into the left plantar in rats,the PWT was measured 3 days,1 day before injection,and 0.5h,1h,2h,1,3,5,and 7 days after injection.（3）To observe the effects of intrathecal AITC on behavior and downstream molecules in rats,successful rats with intrathecal tube were randomly divided into 4groups（n=6）:Sham+Vehicle group,SNI+Vehicle group,SNI+AITC 10μg group and AITC 10μg group,AITC 10μg or vehicle was injected intrathecally pretreatment30 min before surgery,once a day to 7 days after surgery,the PWT was measured 3days,1 day before surgery,and 1,3,5,7 days after surgery.After the last pain test,the spinal cord was swollen in the operation side of the rats,and the expression levels of CaMK2 and CREB were detected by Western Blot.Vehicle:10%DMSO.4 The effect of TRPA1-CaMK2-CREB signaling pathway on neuropathic painThrough the above experiments,the effect of TRPA1 inhibitor and activator on the behavior of rats and the expression of downstream molecules CaMK2 and CREB were obtained.To further study the mechanism of TRPA1 regulating CREB expression by CaMK2,male SD rats were randomly divided into 4 groups（n=3）:Vehicle group,AITC group,AITC+KN-93 group and KN-93 group,intrathecal injection of AITC 10μg pretreatment,and 2 hours later,intrathecal CaMK2 blocker KN-93 50μg or Vehicle,6 hours later,the spinal cords of the rats were taken up,and the expression levels of CREB were detected by Western Blot.Vehicle:10%DMSO.Results1 SNI model induced mechanical pain hypersensitivity in rats and the expression of TRPA1 in ipsilateral spinal dorsal horn of SNI model rats was increased.SNI surgery induced mechanical pain hypersensitivity in rats.Pain behavior test showed that compared with the sham operation group,PWT decreased significantly on the 1st day after SNI and lasted until the end of observation on the 14th day after operation.Western Blot results showed that SNI significantly increased the expression of TRPA1 protein in spinal dorsal horn after operation.The results of immunofluorescence showed that the expression of TRPA1 receptor on the operative side was significantly up-regulated 14 days after SNI,and the up-regulated TRPA1was mainly co-labeled with the neuronal marker NeuN,the astrocyte marker GFAP,and less with the microglia marker IBA1.2 TRPA1 inhibitor HC-030031 can alleviate mechanical hyperalgesia after nerve injury in rats.（1）Intrathecal injection of the TRPA1 inhibitor HC-030031 in rats（intrathecal injection 30 minutes before surgery,once daily until 7 days after surgery）,the pain behavioral test showed that compared with the SNI+Vehicle group,HC-030031dose-dependently increased PWT in rats after SNI,100μg was the best,and 10μg was not obvious.The results indicate that intrathecal application of high dose HC-030031 can inhibit mechanical hyperalgesia induced by SNI surgery.Western Blot results showed that HC-030031 down-regulated the expression of CaMK2 and CREB in the spinal cord of rats after nerve injury.（2）Intrathecal injection of TRPA1 inhibitor HC-030031 100μg（intrathecal injection 7 days after surgery,once daily for 5 days）,the pain behavioral test showed that HC-030031 was significantly higher than the SNI+Vehicle group,rat PWT was increased at 7,9,and 11 days after SNI.The results showed that HC-030031 still attenuated pain in rats after the formation of neuropathic pain.3 TRPA1 activator AITC can induce mechanical hyperalgesia in rats（1）Plantar injection of TRPA1 activator AITC induced acute mechanical hyperalgesia in rats.Pain behavioral tests showed that PWT was significantly reduced 0.5 h after plantar injection of AITC compared with the Vehicle group.There were significant differences in PWT at the other time points except 7 days after injection.AITC 100μg and 300μg plantar injection,PWT no significant difference.（2）Intrathecal injection of HC-030031 100μg can effectively alleviate the mechanical allodynia caused by 300μg of AITC in the plantar injection.The pain behavior test shows that the intrathecal injection of HC-030031 100μg rat PWT in 1,3,and 5 days after AITC 300μg plantar injection was significantly superior to the intrathecal pre-injection of the Vehicle group.（3）Intrathecal injection of AITC 10μg（intrathecal injection 30 minutes before surgery,once daily until 7 days after surgery）caused a gradual decrease in PWT in rats.The results showed that activation of TRPA1 at the spinal cord level caused mechanical hyperalgesia in rats.Western Blot results showed that AITC up-regulated the expression levels of CaMK2 and CREB in the spinal cord of normal rats and rats after nerve injury.4 TRPA1-CaMK2-CREB signaling pathway is involved in the development of neuropathic painWestern Blot results showed that the expression of CREB in AITC+KN-93group was significantly lower than that in AITC group.The results showed that CaMK2 blocker KN-93 can inhibit the expression of CREB induced by AITC.TRPA1 in spinal dorsal horn regulates CREB expression through CaMK2.ConclusionRats Spared nerve injury caused an increase in the expression of TRPA1 in the lumbar spinal cord and may participate in the development of neuropathic pain through the CaMK2-CREB signaling pathway.