The Mechanism Research Of ClC-3 In The Effects Of 17β-estradiol On Pain Of SNI Rats

Object:This study aimed to investigate whether voltage-gated chloride channel-3（ClC-3）on Dorsal Root Ganglion（DRG）neurons is within 17β-estradiol（17β-Estradiol,E₂）plays a role in sciatic nerve injury（SNI）-induced neuropathic pain in ovariectomized（OVX）rats,elucidating the possible mechanism of 17β-estradiol-mediated neuropathic pain in castrated rats.Methods:In this study,female Sprague-Dawley（SD）rats were selected.Two weeks after bilateral ovariectomy.Then the rats were operated on neuropathic pain model（selective injury model of sciatic nerve branch,Spared Nerve Injury,SNI）.Randomly divided into OVX group,OVX+SNI group,OVX+SNI+E₂ group,OVX+SNI+E₂+DMSO（solvent,dimethyl sulfoxid,dimethyl sulfoxide）group,OVX+SNI+E₂+Cltx（ClC-3specific blocker,Chlorotoxin）group.OVX+SNI+E2 rats were subcutaneously injected（s.c.）with 17β-estradiol（30μg/kg/day）once a day for two days after two weeks of OVX for 7 days;OVX+SNI+E₂+Cltx rats received SNI on day 7.From then on,intrathecally（i.t.）Cltx（1μmol/day）was injected for 4 consecutive days.Both OVX and OVX+SNI rats were given intrathecally with Cltx to demonstrate the effect of the blocking agent alone.The acetone cold stimulation and heat radiation stimulation experiments were used to study the noxious behavior of rats.The cold shrinkage duration（PWCD）and thermal shrinkage latency（PWTL）of rats were measured.Immunofluorescence was used to observe DRG of ovariectomized rats Localization of ClC-3 and protein expression levels in ganglia;Western-blot technology was used to monitor the expression of ClC-3 protein in L_4-6-6 DRG,and real-time quantitative polymerase chain reaction（qRT-PCR）was used to detect ClC-3 mRNA Expression change.The whole-cell patch-clamp current clamp technique is used to record the changes in the electrophysiological characteristics of L_4-6-6 DRG cells.Results:There were no changes in behavior and the expression of ClC-3 in rats after 2weeks of ovariectomy.ClC-3 is mainly expressed in small/medium DRG neurons of OVX rats.Compared with OVX rats,SNI of OVX+SNI rats began to show a longer duration of foot contraction to cold stimulation of acetone on the third day,that is,increased sensitivity to cold pain（P<0.001）.Relieve pain after hormone（P<0.001）.Recovery of ClC-3 protein and mRNA expression（P<0.001）.The expression of ClC-3 in DRG neurons was down-regulated after SNI in OVX rats（P<0.001）.Pain was not relieved after combined administration of estrogen and estrogen receptor blocker（P<0.001）.Intrathecal injection of Cltx can inhibit the up-regulation of 17C-estradiol on ClC-3 protein and exacerbate hyperalgesia,but it does not affect the increase of ClC-3 mRNA.Intrathecal injection of Cltx in OVX and OVX+SNI rats increased hyperalgesia and down-regulated ClC-3 protein expression.17β-estradiol reduces DRG neuron excitability caused by SNI in OVX rats,but is blocked by Cltx.Conclusion:（1）Two weeks after ovariectomy,the rats did not show behavioral changes and did not affect the expression of ClC-3 in DRG.（2）ClC-3 is involved in neuropathic pain after SNI in OVX rats.Low-dose continuous administration of 17β-estradiol can relieve hyperalgesia in rats.（3）17β-estradiol may upregulate ClC-3 at the gene level to relieve neuropathic pain,providing a new basis for the treatment of neuropathic pain by estrogen.