米诺环素抑制甲醛炎性痛及机制

Minocycline inhibits formalin-induced inflammatory pain and the underlying mechanism

目的:观察米诺环素对脊髓背角胶状质(substantia gelatinosa,SG)区神经元突触传递的影响,以阐明其在甲醛炎性痛中的作用机制。方法:行为学和免疫组织化学实验:将30只3~5周龄雄性SD大鼠,随机分为对照组(8只)、模型组(8只)、生理盐水模型组(6只)和米诺环素模型组(8只)。对照组采用右后足背皮下注射生理盐水,模型组(甲醛炎性痛模型)采用右后足背皮下注射5%(体积分数)甲醛溶液,生理盐水模型组和米诺环素模型组分别在模型制备前1 h腹腔注射生理盐水和米诺环素。记录4组大鼠足背皮下注射生理盐水或甲醛溶液后1 h内每5 min缩足和舔爪的时间,共记录1 h。痛行为学记录结束1 h后,行4%多聚甲醛心脏灌流取脊髓组织,以免疫组化实验的方法观察脊髓背角c-Fos蛋白的表达。电生理实验:选取26只3~5周龄雄性SD大鼠制作离体脊髓纵切片,每只大鼠随机选取2~5个神经元进行全细胞膜片钳记录,分别记录米诺环素、氟代柠檬酸和多西环素对SG神经元的自发性兴奋性突触后电流(spontaneous excitatory postsynaptic currents,s EPSCs)或自发性抑制性突触后电流(spontaneous inhibitory postsynaptic currents,s IPSCs)的作用。结果:模型组与对照组比较,右侧缩足和舔爪等炎性痛行为及脊髓背角c-Fos蛋白表达显著增加;腹腔注射米诺环素可显著减轻大鼠第二相的炎性痛行为(t=2.957,P<0.05),并减少脊髓背角浅层(Ⅰ~Ⅱ)和深层(Ⅲ~Ⅳ)c-Fos蛋白的表达(t_(Ⅰ-Ⅱ)=3.912,t_(Ⅲ-Ⅳ)=2.630,P<0.05)。米诺环素显著增加SG神经元的s IPSCs的频率至用药前的220%±10%(P<0.05),但对s EPSCs的频率(100%±1%,t=0.112,P=0.951)和幅度(98%±1%,t=0.273,P=0.167)、s IPSCs的幅度(105%±3%,t=0.568,P=0.058)均无显著影响。氟代柠檬酸和多西环素对s IPSCs的频率[分别为:(99%±1%,t=0.366,P=0.099);(102%±1%,t=0.184,P=0.146)]和幅度[分别为:(98%±1%,t=0.208,P=0.253);(99%±1%,t=0.129,P=0.552)]均无显著影响。结论:米诺环素可抑制甲醛炎性痛及减少脊髓背角c-Fos蛋白的表达,这些效应与其增强SG神经元的抑制性突触传递有关,而与其抑制小胶质细胞的激活及抗生素的效应无关。

Objective： To unravel the underlying mechanism of minocycline in formalin-induced inflammatory pain,and to investigate the effects of minocycline on synaptic transmission in substantia gelatinosa （SG） neurons of rat spinal dorsal horn. Methods： Behavioral and immunohistochemistry experiments： 30 male Sprague-Dawley （SD） rats （3-5 weeks old） were randomly assigned to control （n = 8 rats）,model （n = 8 rats）,saline treatment model （n = 6 rats） and minocycline treatment model （n = 8 rats） groups. The control group was subcutaneously injected with normal saline on the right hindpaws.Acute inflammatory pain model was established by injecting 5% （volume fraction） formalin into the right hindpaws. The rats in the latter two groups received intraperitoneal injection of saline and minocycline1 h before the formalin injection,respectively. The time of licking and lifting was recorded every 5 min within 1 h after the subcutaneous injection of normal saline or formalin for all the groups,which was continuously recorded for 1 h. One hour after the pain behavioral recording,the spinal cord tissue was removed following transcardial perfusion of 4% paraformaldehyde. The expression of c-Fos protein in spinal dorsal horn was observed by immunohistochemistry. Electrophysiological experiment： In vitro whole-cell patch-clamp recordings were performed in spinal cord parasagittal slices obtained from 26 male SD rats （3-5 weeks old）. Two to five neurons were randomly selected from each rat for patch-clamp recording.the effects of minocycline,fluorocitrate and doxycycline on spontaneous excitatory postsynaptic currents （s EPSCs） or spontaneous inhibitory postsynaptic currents （s IPSCs） of SG neurons were investigated.Results： Compared with the control group,both the licking and lifting time and the expression of c-Fos protein in ipsilateral spinal dorsal horn of the model group were significantly increased. Intraperitoneal injection of minocycline largely attenuated the second phase of formalin-induced pain responses （t = 2. 957,P〈 0. 05）. Moreover,c-Fos protein expression was also dramatically reduced in both the superficial lamina （Ⅰ-Ⅱ） and deep lamina （Ⅲ-Ⅳ） of spinal dorsal horn （tⅠ-Ⅱ= 3. 912,tⅢ-Ⅳ= 2. 630,P 〈0. 05）. On the other side,bath application of minocycline significantly increased the s IPSCs frequency to220% ± 10% （P〈 0. 05） of the control but did not affect the frequency （100% ± 1%,t = 0. 112,P =0. 951） and amplitude （98% ± 1%,t = 0. 273,P = 0. 167） of s EPSCs and the amplitude （105% ±3%,t = 0. 568,P = 0. 058） of s IPSCs. However,fluorocitrate and doxycycline had no effect on the frequency [ （99% ± 1%,t = 0. 366,P = 0. 099）; （102% ± 1%,t = 0. 184,P = 0. 146）,respectively] and amplitude [（98% ± 1%,t = 0. 208,P = 0. 253）; （99% ± 1%,t = 0. 129,P = 0. 552）,respectively] of s IPSCs. Conclusion： Minocycline can inhibit formalin-induced inflammatory pain and the expression of c-Fos protein in spinal dorsal horn. These effects are probably due to its enhancement in inhibitory synaptic transmission of SG neurons but not its effect on microglial activation or antibiotic action.