Study On The Role Of GPCR-Ca²⁺-CaCCs Signaling Pathway In Neuropathic Pain

Object:To observe the expression of PAR2（protease activated receptor 2）and TMEM16A on DRG（dorsal root ganglion）neurons in the model of chronic constriction injury of the sciatic nerve,and to explore its possible play in the role of pain.To study the concentration changes of Ca²⁺on DRG neural cells in application specific activation of PAR2,SLIGRL-NH2,and to observe the concentration of Ca²⁺on DRG neurons.Intrathecal application of pregabalin on DRG neurons cells of rat in the CCI model to observe the action potential and concentration of Ca²⁺on DRG neurons.Methods:Preparation of CCI rat model,using 37370 automatic heat radiation stimulation to detect the TWL（thermal withdrawal latency）,to detect the expression of PAR2 and TMEM16A on DRG neurons by immunofluorescence,to detect the expression of PAR2 and TMEM16A on DRG neurons in the application of Western blot,to detect the concentration of IP₃ on DRG neurons by ELISA,to detect the concentration of Ca²⁺on DRG neurons with calcium imaging techniques,to detect the action potentials of DRG neurons were by full-cell patch clamp technique.Results:（1）The results of 37370 automatic heat radiation stimulation display,compared to the sham operation group,the difference of TWL in CCI group before surgery was not statistically significant.But the TWL was significantly decreased in CCI group at different time point after operation,and achieve the minimum value in seventh days after surgery,the difference was statistically the significance（P<0.01,n=12）.（2）The immunofluorescence results showed that PAR2 and TMEM16A are co-expressed in DRG neurons were measured.The results showed that compared with the Sham group,the fluorescence intensity on seventh days and fourteenth days of DRG neurons in the PAR2 and TMEM16A increased significantly after operation in CCI group（P<0.01,n=12）,and the difference was statistically significant.（3）The results of Western blot display,compared with the Sham group,protein expression of PAR2 and TMEM16A on seventh days and fourteenth days in DRG neurons were significantly increased after operation in CCI group（P<0.01,n=12）,and protein expression of PAR2 and TMEM16A in CCI group after fourteenth is higher than the CCI group after seventh days（P<0.05,n=12）,and the difference was statistically significant.（4）ELISA technology to detect the IP3 concentration results showed that:compared with Sham group,the concentration of IP₃ on seventh and fourteenth days in DRG neurons increased significantly after operation in CCI group（P<0.01,n=6）,and the difference was statistically significant.（5）Calcium imaging test results show that:compared with Sham group,concentration of Ca²⁺of DRG neurons in CCI group was increased significantly（P<0.01,n=6）.Compared with the CCI group,concentration of Ca²⁺of DRG neurons in pregabalin groupvwas decreased significantly（P<0.05,n=6）.After application of PAR2 specific activation agent SLIGRL-NH2（100μmol/L）10μL on DRG neurons,compared with Sham group,the increasing intensity of calcium ion concentration in DRG neurons in CCI group was more than those in Sham group（P<0.01,n=6）.Compared with CCI group,the calcium concentration of DRG neurons in Pregabalin group showed little change,which was significantly lower than that in CCI group（P<0.05,n=6）.（6）The results of patch clamp technique showed that the threshold of action potential of DRG neurons in CCI group was lower and the frequency was higher than that in sham operation group（P<0.01,n=6）,these results showed that DRG neurons in CCI group were more prone to produce action potential.Compared with the CCI group,the threshold of action potential of DRG neurons in pregabalin group was higher and the frequency was lower than that in CCI group.（P<0.05,n=6）.Conclusions:1.PAR2 and TMEM16A are co-expression on DRG neurons in the rat model of sciatic nerve chronic constriction,their expression and release of IP₃ may lead to chronic constriction injury of the sciatic nerve.2.In the rat model of neuropathic pain on DRG neurons,PAR2 dependence increased concentration of Ca²⁺may be one of the reasons for the formation of neuropathic pain,and pregabalin can reduce PAR2 dependence increased concentration of Ca²⁺.3.The excitability of primary sensory neurons of DRG neurons was increased in the model of chronic compression of sciatic nerve in rats,and pregabalin can reduce its excitability.