臭氧对神经病理性疼痛大鼠脊髓神经元细胞铁死亡的影响

Effects of ozone on iron death in spinal cord neurons of rats with neuropathic pain

目的研究神经病理性疼痛(NP)大鼠模型中是否存在神经细胞的铁死亡,探究O3治疗NP的机制。方法选取60只雄性SD大鼠,随机平均分为3组:神经病理性疼痛模型组(NP组)、假手术组(Sham组)及臭氧组(O3组)。NP、O3组采用坐骨神经结扎术构建神经病理性疼痛模型;Sham组行假手术。O3组术后,在损伤处局部注射15μl O3(40μg/ml),NP和Sham注射等量空气,均为术后1次/d。于术前1 d(T0)及术后1、3、7、14 d(T1~T4)测定模型大鼠的机械缩足反应阈值(MWT)和热缩足潜伏期(TWL)。取大鼠脊髓节段,采用Western Blot检测T1~T4时的谷胱甘肽过氧化物酶4(GPX4)和长链脂酰辅酶A合成酶4(ACSL4)的表达水平,采用免疫荧光技术检测T4时脊髓背角内NeuN+神经元细胞数,采用透射电镜分析T4时脊髓背角内铁死亡特异性改变,采用铁死亡试剂盒检测T1~T4时脊髓背角内铁沉积情况。结果与Sham组相比,NP组和O3组大鼠:T2~T4时MWT降低,TWL缩短;T4时脊髓背角NeuN+神经元数量减少;T4时神经细胞线粒体出现铁死亡特异性改变;T2~T4时神经组织内铁含量增高。与Sham组相比,NP组大鼠GPX4水平降低,ACSL4水平升高。与NP组比较,O3组大鼠:T2~T4时MWT升高,TWL延长;T2~T4时GPX4水平升高,ACSL4水平降低;T4时脊髓背角NeuN+神经元细胞数量增加;T4时神经细胞线粒体萎缩程度改善;T2~T4时神经组织内铁含量降低。上述结果均有统计学意义(均P<0.05)。结论O3可能通过抑制铁死亡的形式,治疗神经病理性疼痛。

Objective To investigate whether ferroptosis of nerve cells exists in a rat model of neuropathic pain(NP),and to explore the mechanism of O3 treatment of NP.Methods Sixty male SD rats were randomly divided into three groups:neuropathic pain model group(NP group),sham operation group(Sham group)and ozone group(O3 group).The partial sciatic nerve ligation was used in the NP and O3 groups to construct a neuropathic pain model.The Sham group was subjected to sham surgery.In the O3 group,15μl of O3(40μg/ml)was injected locally at the injury site,and the NP and Sham were injected with the same amount of air,once per day.The mechanical contraction response threshold(MWT)and thermal contraction latency(TWL)of the rats were measured 1 day before the surgery(T0)and 1,3,7,14 days after the surgery(T1 to T4).The spinal cord segments of rats were collected.The expression levels of glutathione peroxidase 4(GPX4)and long chain fatty acid coenzyme A synthetase 4(ACSL4)at T1 to T4 was detected using Western Blot.The number of NeuN+neuron cells in the spinal dorsal horn at T4 was detected using immunofluorescence technology.The specific changes of ferroptosis at T4 was observed by a transmission electron microscopy.Iron deposition in the spinal dorsal horn at T1 to T4 was measured using ferroptosis kits.Results Compared with the Sham group,rats in the NP group and O3 group showed decreasing of MWT decreased and shortening of TWL at T2 to T4,decreasing of NeuN+neurons in spinal dorsal horn at T4,specific changes of ferroptosis in mitochondria at T4,and increasing of iron content in nerve tissue at T2 to T4.Compared with the Sham group,rats in NP group showed decreasing of GPX4 level and increasing of ACSL4 level.Compared with the NP group,rats in the O3 group showed increasing of MWT and prolonging of TWL at T2 to T4,increasing of the GPX4 level and decreasing of ACSL4 level at T2 to T4,increasing of the number of NeuN+neuron cells in the spinal dorsal horn,improving of the mitochondrial atrophy of nerve cells,and decreasing of the iron content in nerve tissue at T2 to T4.The above results are statistically significant(all P<0.05).Conclusions The mechanism of O3 in treating neuropathic pain may be through inhibition of iron death.