京尼平苷介导Sirt3分子改善大鼠蛛网膜下腔出血神经损伤

Geniposide⁃mediated Sirt3 molecules ameliorate nerve injury in rat subarachnoid haemorrhage

目的探究京尼平苷改善蛛网膜下腔出血的线粒体机制与Sirt3分子相关性。方法40只SD大鼠随机分为假手术组、模型组、烟酰胺核糖组与京尼平苷组,每组10只。假手术组仅对颈正中进行切口并缝合处理,其余组大鼠通过线栓法构建蛛网膜下腔出血模型;假手术组、模型组给予等量的生理盐水,烟酰胺核糖组给予烟酰胺核糖氯酸盐20 mg/(kg·d),京尼平苷组给予京尼平苷30 mg/(kg·d)。神经行为学评分(NSS)评价大鼠神经功能损伤;旷场实验评价认知功能;水迷宫实验评价学习记忆能力。苏木精-伊红(HE)染色观察大鼠脑组织神经细胞损伤情况;ELISA试剂盒检测肿瘤坏死因子-α(TNF⁃α)、γ-干扰素(IFN⁃γ)、转化生长因子-β(TGF⁃β)评价炎症水平;线粒体结构与功能损伤通过电子显微镜与ATP酶活力进行衡定。最后通过Western blot与免疫组化检测大鼠脑组织内Sirt3表达量的改变。结果与假手术组比较,模型组大鼠NSS评分升高(t=23.760),旷场实验中行进距离、中心穿越次数降低(t=14.250、10.720),水迷宫实验中逃避潜伏期增长、穿越平台次数下降(t=4.618、9.238),HE染色结果显示,神经细胞损伤加重(t=4.959),ELISA试剂盒提示促炎因子TNF⁃α、IFN⁃γ表达量升高,抑炎因子TGF⁃β表达量下降(t=4.355,6.716,5.317);电镜与ATP试剂盒提示,线粒体损伤加重,ATP酶活性降低(t=9.882);Western blot与免疫组化提示Sirt3含量降低(t=9.145、7.236),以上差异均有统计学意义(P均<0.05)。与模型组比较,烟酰胺核糖组和京尼平苷组NSS评分降低(t=6.325、3.678),旷场实验中行进距离、中心穿越次数增加(t=4.782、7.324,6.430、4.191),水迷宫实验中逃避潜伏期减少、穿越平台次数增多(t=3.247、6.248,5.145、4.293),损伤神经细胞减少(t=4.272、3.184),TNF⁃α、IFN⁃γ表达量降低、TGF⁃β表达量升高(t=6.824、3.284、5.149,7.145、6.248、4.135),线粒体结构改善,ATP酶活性升高(t=3.241、6.214),Sirt3含量(Western blot与免疫组化)升高(t=5.324、2.241,2.356、4.128),以上差异均有统计意义(P均<0.05)。结论京尼平苷可以通过激活Sirt3分子缓解线粒体炎症损伤,改善蛛网膜下腔出血及行为功能障碍并发症。

Objective To study the mitochondrial mechanism of improvement of subarachnoid haemorrhage by geniposide and the molecular relevance of Sirt3.Methods A total of 40 SD rats were randomly divided into the sham⁃operation group,the model group,the nicotinamide ribose group and the geniposide group,with 10 rats in each group.In the sham⁃operation group,only the middle of the neck was incised and sutured;in other groups,a subarachnoid haemorrhage model was constructed by the thread bolus method;in the sham⁃operation group and the model group,equal amounts of saline were given;in the nicotinamide⁃ribose group,nicotinamide⁃ribose was given at 20 mg/(kg·d),and in the geniposide group,geniposide was given at 30 mg/(kg·d);Neurological severity score(NSS)were used to evaluate the cognitive function of rats;absent field experiment was used to evaluate the cognitive function;water maze experiment was used to evaluate the learning and memory ability;hematoxylin⁃eosin(HE)staining was used to observe the neuronal cell damage in the brain tissue of rats;ELISA kit was used to evaluate the level of inflammation by detecting tumor necrosis factor⁃α(TNF⁃α),interferon⁃γ(IFN⁃γ),transforming growth factor⁃β(TGF⁃β);and the damage to the mitochondrial structure and function was weighed by electron microscopy and ATPase activity.Finally,the alteration of Sirt3 expression in rat brain tissue was determined by Western blot and immunohistochemistry.Results Compared with the sham⁃operated group,the NSS score of rats in the model group was elevated(t=23.760),the distance travelled and the number of centre traversals were reduced in the open field experiments(t=14.250,10.720),the evasion latency was increased and the number of traversals across the platforms was decreased in the water maze experiments(t=4.618,9.238);HE staining results showed that the neuronal cell damage was aggravated(t=4.959);ELISA results suggested that the expression of pro⁃inflammatory factors TNF⁃αand IFN⁃γwas elevated,and the expression of anti⁃inflammatory factor TGF⁃βwas decreased(t=4.355,6.716,5.317);electron microscopy and ATP kit results suggested that the mitochondrial damage was aggravated,and the activity of ATPase was decreased(t=9.882);Western blot and immunohistochemistry results suggested that the content of Sirt3 was reduced(t=9.145,7.236),and all of the above differences were statistically significant(all P<0.05).Compared with the model group,NSS scores were reduced in the nicotinamide ribose and geniposide groups(t=6.325,3.678),the distance travelled and the number of centre traversals were increased in the wilderness field experiments(t=4.782,7.324;6.430,4.191),and the evasion latency was reduced and the number of traversals across the platform was increased in the water maze experiments(t=3.247,6.248;5.145,4.293),fewer damaged neuronal cells(t=4.272,3.184),lower expression of TNF⁃α,IFN⁃γ,and higher expression of TGF⁃β(t=6.824,3.284,5.149;7.145,6.248,4.135),and improvement of mitochondrial structure,increased ATPase activity(t=3.241,6.214)and the amount of Sirt3(Western blot and immunohistochemistry)was elevated in rat brain tissues(t=5.324,2.241;2.356,4.128),and all of the above differences were statistically significant(all P<0.05).Conclusion Geniposide might alleviate mitochondrial inflammatory damage and ameliorate the complications of subarachnoid haemorrhage and behavioral dysfunction by activating Sirt3 molecules.