针刺对脑出血大鼠脑组织神经细胞铁死亡的影响

Effect of Acupuncture on Ferroptosis of Nerve Cells in Rats with ICH

目的:观察针刺对脑出血大鼠脑组织神经细胞铁死亡的影响,探讨针刺促进脑出血大鼠神经功能修复的作用机制。方法:选取健康6周龄SD雄性大鼠96只,随机分为假手术组、模型组、针刺组、抑制剂组,其中每组又随机分为1 d、3 d、7 d 3个亚组,每组8只。模型组采用自体血注入法复制脑出血大鼠模型。假手术组接受与模型组相同的各项操作,微量注射器于相同位置进针,注入等量生理盐水,不注血。针刺组取穴“百会”透“曲鬓”,每日1次。抑制剂组给予肌肉注射去铁胺(DFX)100 mg/kg,每日1次治疗。采用Ludmila Belayev神经功能评分评估各组大鼠神经功能缺损情况。透射电镜观察各组大鼠脑组织神经细胞线粒体形态。免疫组化检测神经元特异性核蛋白(NeuN)表达。丙二醛(MDA)试剂盒检测大鼠脑组织内脂质过氧化物MDA含量。结果:“百会”透“曲鬓”针刺法可以降低脑出血大鼠Ludmila Belayev神经功能评分,减轻脑出血大鼠神经缺损症状。透射电镜观察结果:模型组1 d神经元胞体中可见散在线粒体体积变小,线粒体嵴减少,线粒体外膜破裂。模型组3 d神经元内细胞器明显减少,可见线粒体膜破裂,神经元将近死亡。模型组7 d神经元胞体中线粒体形态有所缓解,但仍可见线粒体体积缩小,线粒体嵴减少和线粒体破裂。针刺组1 d与抑制剂组1 d神经元线粒体以肿胀为主。针刺组3 d与抑制剂组3 d较模型组3d线粒体形态改变均减轻,可见散在线粒体形态变小,同时可见不同程度肿胀的线粒体。针刺组7 d与抑制剂组7 d偶有散在肿胀线粒体。免疫组化结果:与假手术组比较,NeuN阳性细胞数在模型组各时间点均显著减少(P<0.05)。与模型组比较,NeuN阳性细胞数在针刺组及抑制剂组各时间点均显著增多(P<0.05)。MDA测定结果:模型组在出血后各时间点MDA含量均显著升高(P<0.05)。针刺组与抑制剂组大鼠MDA含量较同时间点模型组比较明显减少,结果具有统计学意义(P<0.05)。结论:针刺可能通过抑制脑出血大鼠神经细胞铁死亡促进出血大鼠神经功能修复。

Objective:To observe the effect of acupuncture on ferroptosis of nerve cells in brain tissues of rats with intracerebral hemorrhage(ICH)and to explore the mechanism of acupuncture in promoting restoring the neurological function of ICH rats.Methods:A number of 96 SD male rats were randomly divided into the sham group,the model group,the acupuncture group and the inhibitor group,and each group was again divided into 3 subgroups(1 d,3 d and 7 d),with 8 rats in each subgroup.ICH model was established by injecting autogenous blood.The microinjector was inserted at the same location in the sham operation group without blood injection but normal saline.The acupuncture group was treated with needling Baihui(DU20)through Qubin(GB7)once a day.The inhibitor group was treated with intramuscular injection of deferoxamine(100 mg/kg)once a day.Ludmila Belayev scoring method was used to evaluate the neurological deficit of rats in each group.The morphological changes of mitochondria were observed by transmission electron microscopy(TEM).The expression of NeuN was detected by immunohistochemistry.The content of MDA in brain tissues was detected by MDA Kit.Results:Needling DU20 through GB7 could decrease the Ludmila Belayev score to improve the neurological deficit of ICH in rats.In terms of the TEM results,on 1 d,neuronal soma showed scattered shrunken mitochondria with decreased mitochondrial cristae and rupture of outer mitochondrial membrane in the model group;on 3 d,organelles in neurons were significantly reduced,mitochondrial membrane rupture was observed,and neurons were nearly dead in the model group;on 7 d,the morphological changes of mitochondria in neuronal cell body were improved,but shrunken mitochondria with decreased mitochondrial cristae and rupture of outer mitochondrial membrane could still be seen.Compared to those in the model group,on 1 d,neuronal mitochondria were swollen in the acupuncture group and in the inhibitor group;on 3 d,the morphology of mitochondria was improved,neuronal soma showed scattered shrunken mitochondria,the swollen smitochondrial could be seen at the same time in the acupuncture group and in the inhibitor group;on 7 d,scatted swollen mitochondria could be seen in the acupuncture group and in the inhibitor group.In terms of immunohistochemical results,the number of NeuN positive cells decreased significantly at each time point in the model group compared to that in the sham operation group(P<0.05).Compared to that in the model group,the number of NeuN positive cells increased significantly at each time point in the acupuncture group and in the inhibitor group(P<0.05).In terms of the result of MDA Kit,the content of MDA increased significantly at each time point in the model group compared to that in the sham operation group(P<0.05).Compared to that in the model group,the content of MDA decreased significantly at each time point in the acupuncture group and in the inhibitor group(P<0.05).Conclusion:Acupuncture may promote the repair of nerve function by inhibiting ferroptosis in rats with ICH.