Study On The Role Of TMEM16A In Neuropathic Pain Of SNI Rats

Object: To observe the expression of TMEM16 A,MEK/ERK signaling pathway and NK-1 on DRG(dorsal root ganglion)neurons in the model of spared nerve injury of the sciatic nerve,and to explore the role and possible mechanisms of TMEM16 A played in neuropathic pain.Methods: Adult male Sprague-Dawley rats weighing 180-200 g were randomly divided into 2 groups: Sham group and SNI group.The SNI group used a selective injury of the sciatic nerve branch to establish a neuropathic pain model in rats.Sham group used sham surgery as a control.Firstly,the thermal withdrawal latency(TWL),cold withdrawal duration(CWD)and mechanical withdrawal threshold(MWT)of rats in each group were measured at 1 day before surgery and and 3,7,14 and 21 days after surgery.Then,the surgical side L4-6 DRGs of rats were taken at time points(7,14 and 21 d).The trend of TMEM16 A,MEK/ERK signaling pathway and NK-1 protein expression and cell localization were observed by Western blot and immunofluorescence.Then,the changes of behavior in SNI rats were observed by intrathecal injection of TMEM16 A inhibitor T16Ainh-A01 or MEK inhibitor U0126.Western blot was used to detect TMEM16 A,MEK/ERK signaling pathway and NK-1 expression on DRG in the 14 th day after operation.The changes of behavior in Sham rats were observed by intrathecal injection of TMEM16 A agonist E-act.The changes of MEK/ERK signaling pathway and NK-1 expression in DRG of each group were detected by Western blot.Results:(1)Pain behavioral results showed that compared with Sham group,CWD increased(p<0.001)and MWT decreased(p<0.05)in SNI group,but there was no significant difference in TWL between the two groups(p>0.05).After intrathecal injection of TMEM16 A inhibitor T16Ainh-A01 or MEK inhibitor U0126 in SNI rats,CWD were significantly reduced(p<0.05)and the MWT were significantly increased(p<0.01)from 1 h to 8 h after administration compared with the control group with intrathecal injection of DMSO.After intrathecal injection of TMEM16 A agonist E-act in Sham rats,TWL were significantly reduced(p<0.001),CWD were significantly increased(p<0.05)and the MWT were significantly reduced(p<0.01)from 1 h to 8 h after administration,compared with the control group with intrathecal injection of DMSO;(2)Immunofluorescence results showed that TMEM16 A,p-ERK1/2 and NK-1 were mainly expressed on pain-related neurons;(3)Western blot showed that compared with Sham group,the expression of TMEM16 A,p-MEK,p-ERK1/2 and NK-1 increased after SNI,and the expression was highest on the 14 th day(p<0.01)after SNI.After intrathecal injection of T16Ainh-A01 or U0126 in SNI rats,the expression of p-MEK,p-ERK1/2 and NK-1 was decreased(p<0.01),but injection of U0126 did not inhibit the increase of TMEM16A(p>0.05).After intrathecal injection of E-act in Sham rats,p-MEK,p-ERK1/2 and NK-1 were all increased in expression compared with the DMSO-injected control group(p< 0.001).Conclusion:(1)TMEM16A is involved in neuropathic pain in SNI rats.TMEM16 A inhibitor T16Ainh-A01 can significantly alleviate hyperalgesia in SNI rats;(2)MEK/ERK signaling pathway isinvolved in neuropathic pain in SNI rats.MEK inhibitor U0126 can significantly alleviate hyperalgesia in SNI rats;(3)TMEM16A regulates the expression of NK-1 through MEK/ERK signaling pathway and participates in neuropathic pain in SNI rats.