天麻素对蛛网膜下腔出血模型大鼠Sirt6/FoxO3a通路及神经功能损害的影响

Effects of gastrodin on Sirt6/FoxO3apathway and neurological impairment in rats with subarachnoid hemorrhage

目的探讨天麻素对蛛网膜下腔出血(Subarachnoid hemorrhage,SAH)模型大鼠沉默信息调节因子6(Silent information regulator 6,Sirt6)/叉头转录因子3a(Forkhead transcription factor 3a,FoxO3a)通路相关蛋白表达水平及神经功能的影响。方法将SD(Sprague Dawley,SD)大鼠随机分为假手术(Sham)组、模型(SAH)组、Sirt6抑制剂(OSS128167)组(尾静脉注射,100μg/只)、天麻素组(腹腔注射,100 mg/kg)、天麻素+OSS128167组(100 mg/kg+100μg/只);除Sham组外,其余各组均采用2次枕大池注血法建立SAH模型;各组分别于造模成功后开始给药,1次/d,共7 d,末次给药12 h后Garcia神经功能评分法评估脑神经功能受损程度;烘干法检测脑组织含水量;苏木精-伊红染色(Hematoxylin and eosin staining,HE)检测各组大鼠脑组织神经元损伤程度;酶联免疫吸附法(Enzyme-linked immunosorbent assay,ELISA)检测脑组织紧密连接相关蛋白1(Zonula occluden-1,ZO-1)和血脑屏障闭锁蛋白(Occludin)水平;蛋白免疫印迹法(Western blot,WB)检测脑组织通路蛋白Sirt6,FoxO3a、磷酸化FoxO3a(Phosphorylated FoxO3a,p-FoxO3a)、类维生素A X受体(Retinoid AX receptor,RXR)、白细胞介素6(Interleukin-6,IL-6)、肿瘤坏死因子-α(Tumor necrosis factor alpha,TNF-α)相对表达水平。结果与Sham组比较,SAH组大鼠神经元变性坏死,神经功能评分、ZO-1及Occludin水平、RXR,Sirt6蛋白相对表达水平降低(P<0.05),脑组织含水量、p-FoxO3a/FoxO3a,IL-6,TNF-α蛋白相对表达水平升高(P<0.05)。与SAH组比较,OSS128167组大鼠神经元变性坏死进一步加重,神经功能评分、ZO-1及Occludin水平、RXR,Sirt6蛋白相对表达水平降低(P<0.05),脑组织含水量、p-FoxO3a/FoxO3a,IL-6,TNF-α蛋白相对表达水平升高(P<0.05);天麻素组大鼠神经元变性坏死现象缓解,神经功能评分、ZO-1及Occludin水平、RXR,Sirt6蛋白相对表达水平升高(P<0.05),脑组织含水量、p-FoxO3a/FoxO3a,IL-6,TNF-α蛋白相对表达水平降低(P<0.05);天麻素+OSS128167组大鼠上述指标水平变化与天麻素组相反,且有明显差异(P<0.05)。结论天麻素可促进RXR/Sirt6/FoxO3a通路活化,减轻SAH模型大鼠神经功能损伤。

Objective To investigate the effects of gastrodin on the expression of silent information regulator 6(Sirt6)/forkhead transcription factor 3 a(FoxO3 a) pathway related proteins and neruologicaldysfunction in rats with subarachnoid hemorrhage(SAH). Methods SD rats were randomly divided into sham operation group(Sham), model group(SAH), SIRT6 inhibitor(OSS128167) group(tail vein injection, 100 μg/mouse), gastrodin group(intraperitoneal injection, 100 mg/kg), gastrodia Supplement+OSS128167 group(100 mg/kg+100 μg/head), except for the Sham group, all the other groups used the second cistern magna blood injection method to establish SAH model, each group started to take the medicine after successful modeling, once per d, a total of 7 d. Twelve hours after the last administration, the Garcia neurological function score method was used to assess the degree of neurological damage, the drying method was used to detect the water content of the brain tissue, and the hematoxylin-eosin staining(HE) was used to detect the degree of neuronal damage in the brain tissue of each group. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of zonula occluden-1(ZO-1) and blood-brain barrier lock protein(occludin). Western blotting was used to detect the expression of Sirt6, FoxO3 a, and phosphorylated FoxO3 a(p-FoxO3 a), retinoid AX receptor(RXR), interleukin 6(IL-6), and tumor necrosis factor α(TNF-α). Results Compared with the Sham group, the neuronal cell degeneration and necrosis, neurological function score, ZO-1 and occludin levels, RXR, Sirt6 protein expression in the SAH group were decreased(P<0.05), and brain water content, expressions of p-FoxO3 a/FoxO3 a, IL-6, TNF-α proteins were increased(P<0.05). Compared with SAH group, neuronal cell degeneration and necrosis in OSS128167 group was further aggravated, neurological function score, expressions of ZO-1 and occludin, RXR, Sirt6 proteins in the OSS128167 group were decreased(P<0.05), brain water content, expressions of p-FoxO3 a/FoxO3 a, IL-6, and TNF-α in the OSS128167 group were increased(P<0.05). The degeneration and necrosis of neuronal cells in the gastrodin group alleviated, the nerve Functional score, expressions of ZO-1 and occludin levels, RXR, Sirt6 proteins in the gastrodin group were increased(P<0.05), brain water content, expressions of p-FoxO3 a/FoxO3 a, IL-6, TNF-αin the gastrodin group were decreased(P<0.05), comparing to the SAH group. The changes in the above indexes of the rats in the Gastrodin+OSS128167 group were opposite to those in the Gastrodin group and the difference was statistically significant(P<0.05). Conclusion Gastrodin could promote the activation of RXR/Sirt6/FoxO3 a pathway and improve the neurological damage in SAH model rats.