Facilitation Of MrgprD By TRPA1 Participates In Neuropathic Pain

Background:Neuropathic pain is defined as damage to the somatosensory system or pain caused by the disease.This disease has a serious impact on the patient’s life and health.However,the mechanism of neuropathic pain is still unclear.so there is a lack of effective therapeutic drugs.Mrgs(Mas-related genes)related receptors are a class of novel G protein-coupled receptors(GPCRs)closely related to pain,and MrgprD is one of the subtypes that plays an important role in the perception and regulation of pain.Previous studies have generally assumed that G protein-coupled receptors are often involved in pain production along with downstream TRP channels.We speculate that MrgprD is involved in the production of neuropathic pain.Thus,we conducted research related neuropathic pain and its downstream pathways if MrgprD participationPurposes:1.To study whether MrgprD is involved in neuropathic pain;2.To explore the downstream pathway of MrgprD-mediated neuropathic pain.Methods:(1)After the model of chronic constriction(CCI)of wild-type(WT)mice and MrgprD-/-mice,by mouse ethology experiment,the differences of mechanical pain,cold pain,thermal pain in mice were detected.It was judged from the overall behavioral theory whether MrgprD was involved in the production of neuropathic pain.(2)The expression of MrgprD in MrgprD-/-mice(MrgprD substituted by GFP)control group,sham-operated group and CCI-modeled group L4 and L5 DRG neurons was detected by immunofluorescence staining;and the mRNA levels of MrgprD in WT mice after CCI model was observed by Realtime-PCR;calcium imaging experiments were performed to determine the response of DRG neurons and MrgprD-specific agonist β-alanine in CCI model mice.(3)Calcium imaging was used to observe the co-reaction of MrgprD agonist β-alanine with TRPA1 agonist mustard oil and DRG neurons.The immunofluorescence double staining method was used to detect the co-expression of MrgprD and TRPA1 in DRG neurons,and the relationship between MrgprD and TRPA1 in DRG neurons was determined morphologically.(4)Calcium imaging method to detect whether TRPA1 blocker HC-030031 inhibits β-alanine-induced calcium influx in DRG neurons;electrophysiological experiments examined whether HC-030031 inhibitsβ-alanine-induced inward currents.(5)To further verify the relationship between MrgprD and TRPAl,we compared the DRG neurons and β-alanine responses in WT and TRPA1-/-mice with calcium imaging.At the same time,the responses of DRG neurons in the WT mice and MrgprD-/-mice and the TRPA1 agonist mustard oil were also observed.(6)The relationship between TRPA1 and MrgprD was verified by HEK293 cells and P-alanine reactions transfected with MrgprD and TRPA1 plasmids.(7)It is generally believed that downstream of G-protein-coupled receptors mediates signal transduction through PKA,PKC,or PLC.We used PKA and PKC and PLC inhibitors to determine the downstream pathway of MrgprD through calcium imaging experiments.(8)In past studies,subcutaneous injection of P-alanine to activate MrgprD can cause pruritus.We observed the change in pain perception(paw behavior)and pruritus(claw behavior)in the CCI model by subcutaneous injection of beta-alanine.Results:(1)After CCI was established,compared with WT mice,there was a significant difference in mechanical pain behavior and cold pain behavior on the 7th day in MrgprD-/-mice,but there was no difference in thermal pain,showing that MrgprD is involved in the development of mechanical pain and cold pain in neuropathic pain.(2)Immunofluorescence experiments revealed that the expression of MrgprD-positive neurons in CCI-model mice was significantly increased.Realtime-PCR showed that the mRNA expression of MrgprD in CCI mice was significantly increased compared with the control group.Calcium imaging experiments found that the DRG neurons in CCI surgery group were significantly more sensitive to β-alanine than the control group.We believe that the expression of MrgprD is significantly increased under neuropathic pain conditions.(3)Calcium imaging experiments found that β-alanine-activated DRG neurons can be activated by mustard oil.Immunofluorescence experiments revealed that most of the MrgprD-positive neurons express TRPA1.(4)Calcium imaging results showed that the TRPA1 inhibitor HC-030031 significantly inhibited P-alanine-induced calcium influx.Whole-cell patch clamp experiments confirmed that the TRPA1 inhibitor HC-030031 significantly inhibited β-alanine-induced inward currents.(5)Compared with WT mice,the response size and cell number of both DRG neurons and P-alanine in TRPA1-/-mice were significantly decreased;however,there was no difference in the response of MrgprD-/-mouse DRG neurons and TRPA1 agonist mustard oil to WT mice.(6)The response of HEK293 cells and P-alanine co-transfected with MrgprD and TRPA1 was significantly greater than that of HEK293 cells transfected with MrgprD alone.P-alanine does not react with untransfected HEK293 cells.(7)Inhibitors of PKA significantly inhibited calcium influx induced byβ-alanine-activated MrgprD in DRG neurons,and inhibitory effects of PKC and PLC blockers were not significant.At the same time,PKA blockers also significantly inhibited calcium influx induced by β-alanine activation of MrgprD on HEK293 cells co-transfected with MrgprD and TRPA1.We have detected the activation of β-alanine DRG neurons induced phosphorylation TRPA1.Such phosphorylation can be inhibited by the PKA blocker.(8)β-alanine injection at the same time can cause pain and itch.In the CCI model,the pain sensation obviously increases,but the itchiness does not change.In neuropathic pain conditions,the pain perception was significantly reduced in MrgprD-/-mice and WT mice.Compared with WT mice,the pain behavior induced by foot injection of β-alanine in TRPA1-/-mice was decreased,but the pain behavior of TRPAl-/-mice decreased more significantly in the CCI model.Conclusion:MrgprD is involved in the production of neuropathic pain as a member of the Mrgs family.We further found that MrgprD mainly participates in the production of neuropathic pain through the PKA-TRPA1 pathway.In the condition of neuropathic pain,plantar injection of β-alanine mainly causes painful behavior.Significance of research:The study of MrgprD involvement in neuropathic pain explains the new mechanism of neuropathic pain and provides a new target for the development of related analgesic drugs.